Structure-tuned membrane active Ir-complexed oligoarginine overcomes cancer cell drug resistance and triggers immune responses in mice

The development of chemotherapy, an important cancer treatment modality, is hindered by the frequently found drug-resistance phenomenon. Meanwhile, researchers have been enthused lately by the synergistic use of chemotherapy with emerging immunotherapeutic treatments. In an effort to address both of the two unmet needs, reported herein is a study on a series of membrane active iridium(iii) complexed oligoarginine peptides with a new cell death mechanism capable of overcoming drug resistance as well as stimulating immunological responses. A systematic structure–activity relationship study elucidated the interdependent effects of three structural factors, i.e., hydrophobicity, topology and cationicity, on the regulation of the cytotoxicity of the Ir(iii)-oligoarginine peptides. With the most prominent toxicities, Ir-complexed octaarginines (R8) were found to display a progressive oncotic cell death featuring cell membrane-penetration and eruptive cytoplasmic content release. Consequently, this membrane-centric death mechanism showed promising potential in overcoming multiple chemical drug-resistance of cancer cells. More interestingly, the eruptive mode of cell death proved to be immunogenic by stimulating the dendritic cell maturation and inflammatory factor accumulation in mice tumours. Taking these mechanisms together, this work demonstrates that membrane active compounds may become the next generation chemotherapeutics because of their combined advantages.


Structure-tuned Membrane Active Ir-complexed Oligoarginine Overcomes Cancer Cell Drug Resistance and Triggers Immune Responses in Mice
Shuangshuang Ji a,b , Xiuzhu Yang b , Xiaolong Chen a,b , Ang Li a,b , Doudou Yan c , Haiyan Xu c , Hao Fei a,b* [a] School of Nano-Tech and Nano-Bionics, University of Science and Technology of China, Hefei, 230026, P R China.
[b] CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, 215123, P R China.
[c] Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100005, P R China. Table S1.

Standard curves of Ir-peptides
The coordinated Ir-peptides were diluted for various concentrations (1, 5, 10, 25, 50 μM) in Dulbecco's modified Eagle medium (DMEM) and incubated at 37 o C for 24 h, then the samples were excited by 328 nm to get the emission spectra, Ex Slit:5.0 nm, Em Slit:10.0 nm,700 V. The fluorescence intensity at 520 nm was calculated to establish the standard curves. (F-4600, HITACHI)

Coordination efficiency and hydrophobicity of peptides
Peptides with or without coordination were filtered by 220 nm filter and transferred to autosampler vials and separated on an UPLC by ACQUITY UPLC C18 BEH 1.7 μm, 50 mm × 2.1 mm column using an Acquity UPLC system. (Waters Corp., USA) The gradient mobile phase consisted of acetonitrile (A) and H 2 O (B) both containing 0.1% trifluoroacetic acid. A typical 15 min sample run consisted of 2 min of 5% solvent A and 95% solvent B followed by an increment of solvent A up to 90% for the remaining 6 min. Then the proportion was held for 1 min and changed to 5% solvent A and 95% solvent B in the next 4 min, and held for 2 min. The analytes were monitored by a photo diode array (PDA) detector set at 220 nm and 254 nm and the flow rate was set to 0.3 mL min -1 .

Separation of isomer
Ir-cR5 was filtered and transferred to an autosampler vial and separated on an ACQUITY UPLC BEH Shield RP18 1.7 μm, 100 mm × 2.1 mm column. (Waters Corp., USA) The gradient mobile phase consisted of acetonitrile (A) and H 2 O (B) both containing 0.1% trifluoroacetic acid. A typical 15 min sample run consisted of 2 min of 20% solvent A and 80% solvent B followed by an increment of solvent A up to 23% for the remaining 2 min. Then the proportion of solvent A was increased to 45% in the following 8 min and decreased to 20% in the last 3 min. The analytes were monitored by a photo diode array (PDA) detector set at 220 nm and 254 nm and the flow rate was set to 0.3 mL min -1 . The separated metallopeptides were collected to lyophilized and quantified by ICP-MASS, then for MTT assay.

Circular dichroism assay of Ir-peptides
Ir-aRn (n=3, 5, 7), Ir-cR3 and Ir-cR7 were collected by the UPLC method described above to remove DMSO. The collected Irpeptides were lyophilized and quantified by ICP-MASS. The Ir-peptides, isomers, mixture of two isomers were diluted to same concentration for circular dichroism test. (Chirascan-plus, Applied photophysics)

LogP assay
Water and octanol (1:1, v/v) were saturated for 24 h at room temperature and separated as aqueous phase and oil phase. Ir-cRn or Ir-aRn were added in the tube containing 1 mL aqueous phase and 1 mL oil phase with a final concentration of 20 μM, Ir-cRn or Ir-aRn in 2 mL aqueous phase (40 μM) were used to establish standard curves. Samples were stirred for 24 h at room temperature, and groups only containing aqueous phase were diluted to various concentrations (1, 5, 10, 20, 40 μM) to build the standard curves by fluorescence spectrophotometer, Ex Slit:5.0 nm, Em Slit:5.0 nm, 650 V. Then the aqueous phase's fluorescence intensities in samples containing both oil and aqueous phases were scanned, the concentration in aqueous phase of each Ir-peptide was calculated by standard curve, the Ir-peptide's initial concentration in aqueous phase minus 24 hour's concentration in aqueous phase to obtain the 24 hour's concentration in oil phase, logP=log(c oil /c aqueous ).

Induction of cisplatin resistant A549/DDP cells
A549 cells were incubated in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum, 37 o C, 5%CO 2 until entering the logarithmic growth period. Then the medium was replaced by DMEM medium containing 10% fetal bovine serum and 0.125 μM cisplatin. The cells were incubated with cisplatin for 24 h and the medium was removed and no cisplatin DMEM medium containing 10% fetal bovine serum was added until the next logarithmic growth period. The induction followed this cycle until the concentration of cisplatin reached 4 μM. The concentration of cisplatin was twice as much as before in each induction. Then the cells were incubated with DMEM containing 10% fetal bovine serum and 4 μM cisplatin for two months and medium containing cisplatin was replaced by no-cisplatin medium two weeks before experiment.

Cellular uptake of Ir-peptides
HeLa cells were cultured in 6-well microplates, 3*10^5 cells per well overnight. Ir-cRn, Ir-aRn at the concentration of 1/2 IC 50 μM were incubated with HeLa cells for 24 hours. The fluorescence intensity at 520 nm of supernatant after 24 h incubation was measured. The uptake ratios were calculated based on the fluorescence standard curves established above.

Subcellular colocalization of Ir-cR8
HeLa cells were cultured in glass-bottomed well overnight, 3*10^5 cells per well. After washing with PBS, organelles including lysosomes, mitochondria, endoplasmic reticulum were stained by LysoTracker Red DND-99, Mito-TrackerRed FM, ER-TrackerTM Red (the working concentrations follow the instructions) respectively for 30 min, then the dyes were replaced by DMEM containing 5 μM Ir-cR8 for another 10 min incubation before observed by CLSM. (OLYMPUS, FV3000)

Cellular ROS analysis
HeLa cells were cultured in 6-well microplate, 3*10^5 cells per well overnight. After washing, PBS, Ir-cR8 at its IC 50 concentration were incubated with cells for 0.5, 1 h. CellRox Deep Red (Thermo Fisher, USA), a fluorogenic probe for measuring cellular oxidative stress in live or fixed cell was then added for another 30 min incubation (the working concentration follow the instruction). Next, cells were digested and washed three times by PBS. The obtained cells were subjected to flow cytometry analysis. (Accuri C6, Bio-Rad)

Endocytosis inhibitor Assays
HeLa cells were seeded in 6-well microplate overnight, 3*10^5 cells per well overnight. Endocytosis inhibitor chlorpromazine (5.0 μg mL -1 ) was pre-incubated with cells for 1 h. Ir-cR8 (5 μM) was added in each well and incubated at 37 o C for 2 h except the 4 o C group which was placed at 4 o C for 2 h. Then the fluorescence intensity at 520 nm of Ir-cR8 in supernatant of each group was scanned and calculated by the standard curves built above. HeLa cells were cultured in glass-bottomed well for overnight, 3*10^5 cells per well. After washing with PBS, 250 μg mL -1 Rhb-Dextran and 5 μM Ir-cR8 were added in the well and incubated with Hela cells for 1 h, then the cells were observed by confocal laser scanning microscope (CLSM). (OLYMPUS, FV3000)

Hemolysis analysis
Erythrocytes 1.4*10^7 per well were incubated with Ir-aRn, Ir-cRn (n=4, 6, 8), RL1, RL2 of various concentrations for 2 h, water was used as positive control and isotonic phosphate buffered solution was used as negative control. Then the samples were shaked at the speed of 87 rpm under 37 o C and centrifuged at 3000 rpm for 5 min, the absorbance at 405 nm of supernatant was obtained by microplate spectrophotometer (Victor X4, PerkinElmer).

ROS inhibition assay
NAC is an ROS inhibitor. Cells of the ROS inhibition group were pre-incubated with NAC (10 mM) for 1 h. Afterwards, PBS, Ir-cR8 (7, 10 μM) were added into the wells with or without pre-incubation of NAC for 1 h. Cell viability at 15, 30, 45, 60 min or 24 h of each group was calculated by MTT assay.

Calpain inhibition assay
E64 is an irreversible calpain inhibitor. Cells of the calpain inhibition group were pre-incubated with E64 (15 μM) for 1h.

S5
Afterwards, PBS, Ir-cR8 (7, 10 μM) were added into the wells with or without pre-incubation of E64 for 1 h. Cell viability at 15, 30, 45, 60 min or 24 h of each group was calculated by MTT assay.

Caspase activity/inhibition analysis
Ir-cRn (n=4, 6, 8) were incubated with HeLa cells for 0.5, 1 h at their IC 50 concentrations respectively. Cells incubated with DMEM or staurosporine (1 μM) for 4 h was used as negative or positive control. 100 μL Caspase-Glo® 3/7 reagent (Promega, USA) was added into each well and incubated for 2 h. luminescence(RLU)intensity of each well was analyzed by microplate spectrophotometer (Victor X4, PerkinElmer). Z-VAD-FMK is an inhibitor for caspase family. PBS, Ir-cR8 (7, 10 μM) were added to the wells with or without pre-incubation of Z-VAD-FMK (50 μM) for 1 h. The incubation time for short period MTT assay were 15, 30, 45, 60 min, for long time period incubation was 24 h. MTT was added into each well after incubation. The absorbance at 490 nm at each well was analyzed by microplate spectrophotometer (Victor X4, PerkinElmer).

Pyroptosis inhibition assay
HeLa cells (8*10^3 per well) were seeded in 96-well microplate for 12 h. For the granzyme A mediated pyroptosis inhibition groups, 3,4-dichloroisocoumarin (DIC, 10 μM) was pre-incubated with HeLa cells for 1 h. Then, PBS, Ir-cR8 (7, 10 μM) were added in wells with/without DIC and incubated for short or long period as mentioned above. Afterwards, MTT was added for 4 h incubation and then medium were replaced by DMSO. The absorbance at 490 nm was scanned by microplate spectrophotometer (Victor X4, PerkinElmer).

Ferroptosis inhibition assay
HeLa cells (8*10^3 per well) were seeded in 96-well microplate for 12 h. For the ferroptosis inhibition groups, ferrostatin-1 (5 μM) was pre-incubated with HeLa cells for 1 h. Then, PBS, Ir-cR8 (7, 10 μM) were added in wells with/without Ferrostatin-1 and incubated for short or long period as mentioned above. Afterwards, MTT was added for 4 h incubation and then medium were replaced by DMSO. The absorbance at 490 nm was scanned by microplate spectrophotometer (Victor X4, PerkinElmer).

Necroptosis inhibition assay
HeLa cells (8*10^3 per well) were seeded in 96-well microplate for 12 h. For the necroptosis inhibition groups, necrostatin-1 (40 μM) was pre-incubated with HeLa cells for 1 h. Then, PBS, Ir-cR8 (7, 10 μM) were added in wells with/without Necrostatin-1 and incubated for short or long period as mentioned above. Afterwards, MTT were added for 4 h incubation and then medium were replaced by DMSO. The absorbance at 490 nm was scanned by microplate spectrophotometer (Victor X4, PerkinElmer).

In vitro DC maturation
Dendritic cells were isolated from the bone marrow of 6-week-old BALB/c mice purchased from Changzhou Cavens Lab Animal Co. Ltd. and incubated with DMEM medium containing 10% FBS, 100U/mL penicillin-streptomycin, 20 ng/mL murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and 5 ng/ml IL-4. 4T1 cells were incubated with PBS, Ir-cR8, cisplatin and the supernatant of each group was added into dendritic cells for 24 h incubation. The DC were collected and stained with anti-CD11c-FITC, anti-CD80-PE, anti-CD86-APC for flow cytometer analysis.

Animal welfare
This study was performed in strict accordance with the policy published in China State Council Gazette Supplement (Aug 20, 2017) "Regulations on Administration of Animals Used as Subjects of Experiments" and was approved by the Institutional Animal Care and Use Committee of Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences.