Phosphorescent cationic iridium(iii) complexes dynamically bound to cyclodextrin vesicles: applications in live cell imaging

Cyclodextrin vesicles decorated with luminescent Ir(iii)-complexes are used as contrast agents for cell imaging.


Mammalian expression vectors:
The mRFP-Mito construct was obtained by exchanging the fluorescent tag of pmTurquoise2-Mito with mRFP from mRFP-C1 using restriction enzymes AgeI (NEB, R3552L) and XhoI (NEB, R0146L). mRFP1-C1 was a gift from Robert Campbell, Michael Davidson and Roger Tsien (Addgene plasmid # 54764). pmTurquoise2-Mito was a gift from Dorus Gadella (Addgene plasmid # 36208). 6 mRFP-Rab7 was a gift from Ari Helenius (Addgene plasmid # 14436). 7 Live cell microscopy: Live-cell imaging employed a LSM 780 confocal laser scanning microscope (CLSM, Carl Zeiss) equipped with the objective lense Plan-Apochromat x 63/1.4 oil and differential interface contrast objective lenses (Carl Zeiss) in a 37°C environment with 5 % CO 2 . Data was processed using Fiji. 8 For analysis of uptake efficiency, confocal pictures of single cells were thresholded for excitation at 405 nm (emission collected between 410 and 556 nm) and the integrated signal density (mean fluorescence value * area) was calculated for the thresholded area. Statistics were done using the Mann-Whitney U test. Artificial colors (cyan and red) were used for presentation of the data.
Uptake experiments: For uptake experiments, freshly prepared Ir-complexes and CDV in PBS (5 µM and 15 µM respectively) were diluted in mixed medium to final concentrations of 1.25 µM and 3.75 µM, respectively, and added to HUVECs. Uptake of the complex was monitored for 90 min in a live cell experimental setup (37°C and 5 % CO 2 ) using CLSM.
Cytotoxicity assay: Toxicity of CDV, 1b and 1b@CDV pre-solved in assay medium or 1b dissolved in DMSO on HUVECs was assessed by measuring the lactate dehydrogenase (LDH) activity released from damaged cells. The cells were seeded in 96well plates with density of 1500 cells/well and cultured for 2 d. Subsequently, the culture medium was replaced with assay medium containing different concentrations, of the compounds ranging from 0 to 100 µM. Thereafter, the plates were left in the incubator for 2h at 37°C. The maximal LDH activity that represents the control values was determined by addition of 1% Triton X-100 for 10 min to completely lyse the cells. After transfer of the cell supernatants to a new 96-well plate, they were mixed in a volume ratio of 1:1 with reaction mixtures containing the tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5phenyl-2H-tetrazolium (INT), followed by incubation for exact 30 min at 37 C by protected from light. The reaction was stopped by addition of HCl (1 N) to each well. The values of relative toxicity (percentage of LDH activity as compared to the maximal value obtained after Triton treatment) were calculated employing the equation 1 after measuring the absorbance at 492 nm on a Microplate Reader. The concentration-response curve obtained was fitted to a growth-sigmoidal function. LDH assays were carried out for each sample in sextuplicate (n = 6).          ratio -CD/1b Zeta-potential:

4-(But-3-yn-1-yl)-4`-methyl-2,2`-bipyridine (5)
All flasks have to be dried and this reaction was conducted in nitrogen. 4,4'-dimethyl-2,2'-bipyridine (465 mg, 2.52 mmol, 1.0 equiv.) was dissolved in dry THF (25 ml) and cooled to -78 o C for 15 minutes (dry ice and acetone bath). Another flask containing 5 mL THF and distilled diisopropylamine (0.43 ml, 3.10 mmol, 1.2 equiv.) was cooled to -78 o C, and n-butyllithium (1.59 ml, 2.5 M, 2.54 mmol, 1.0 equiv.) was added dropwise stirring for 15 minutes to prepare the lithium diisopropylamide (LDA). The freshly prepared LDA solution was added dropwise via a cannula to the THF solution of 4,4'-dimethyl-2,2bipyridine, and the solution was stirred for 1h. The (3-bromoprop-1-yn-1-yl)trimethylsilane (0.5 mL, 3.53 mmol, 1.4 equiv.) was dissolved in THF (3 mL), cooled to -78 o C and added dropwise via a cannula to the 4,4'-dimethyl-2,2-bipyridine solution. The solution was stirred at this temperature for 0.5 h before being allowed to warm up to room temperature, and then stirred overnight. The mixture was poured into distilled water and extracted three times with DCM. The organic layer was collected and dried with Na 2 SO 4 . Filtration and evaporation under reduced pressure gave the crude product. The crude product was purified by column chromatography (silica, Hexane/ethyl acetate= 3:1, Rf: 0.38) to give a colourless solid which was then redissolved in methanol (20 mL) with K 2 CO 3 (600 mg, 4.34 mmol, 1.7 equiv.) and stirred for 4 hours at room temperature. DCM and water were used for three times extraction after removing the methanol. The organic fractions were combined, washed with a portion of brine and dried over Na 2 SO 4 . Filtration and evaporation under reduced pressure gave the crude product. This solid was kept in vacuum for 1 h to remove remaining TMS and obtain the N^N ligand 5 as white solid. (160 mg, 0.72 mmol, 28 %).

General procedure 1: Synthesis of [Ir(C^N) 2 ] 2 Cl 2 dimers
Ir dimers were synthesized following the procedure of Nanoyama 14 . The corresponding ligand (2.2 equiv.) and IrCl 3 . 3H 2 O (1.0 equiv.) were dissolved in 2-ethoxyethanol to give a concentration of 0.02 M. The mixture was degassed by multiple vacuum and N 2 purging cycles. Afterwards, it was refluxed at 140°C for overnight, cooled to room temperature and the precipitate was filtered and washed with water, hexane and diethyl ether. The product was used without any further purification.

General procedure 2: Synthesis of [Ir(C^N) 2 (N^N)]Cl complexes
[Ir(C^N) 2 ] 2 Cl 2 dimer and the ancillary N^N ligand (2.5 equiv.) were added to a Schleck tube containing a mixture of DCM and MeOH. The mixture was degassed by multiple vacuum and N 2 purging cycles. Afterwards, it was stirred at 55 °C for 19 h under nitrogen atmosphere and cooled to room temperature. The solvent was evaporated under vacuum and the product was purified by flash column chromatography.

General procedure 3: Synthesis of [Ir(C^N) 2 -Ad]Cl complexes
CuSO 4 and L-ascorbic acid were dissolved in a mixture of H 2 O and MeOH (1:1). The desired alkyne compound (1.0 equiv.) was added and the solution was vigorously stirred. After the addition of the corresponding azide (1.1/2.4 equiv.) and pyridine (5.0/10.0 equiv.), the solution was stirred for 48 hours at room temperature. The solvent was evaporated, and the product was purified by column chromatography.