Nucleus‐Targeted Organoiridium–Albumin Conjugate for Photodynamic Cancer Therapy

Abstract An organoiridium–albumin bioconjugate (Ir1‐HSA) was synthesized by reaction of a pendant maleimide ligand with human serum albumin. The phosphorescence of Ir1‐HSA was enhanced significantly compared to parent complex Ir1. The long phosphorescence lifetime and high 1O2 quantum yield of Ir1‐HSA are highly favorable properties for photodynamic therapy. Ir1‐HSA mainly accumulated in the nucleus of living cancer cells and showed remarkable photocytotoxicity against a range of cancer cell lines and tumor spheroids (light IC50; 0.8–5 μm, photo‐cytotoxicity index PI=40–60), while remaining non‐toxic to normal cells and normal cell spheroids, even after photo‐irradiation. This nucleus‐targeting organoiridium‐albumin is a strong candidate photosensitizer for anticancer photodynamic therapy.

Determination of free thiol content of HSA.

Cellular localization assay.
Immunofluorescence staining of HSA in A549 cells.
Detection of intracellular 1 O2.            and human normal liver cell line (LO2) were purchased from Sigma-Aldrich. They were cultured in Roswell Park Memorial Institute medium (RPMI-1640, without phenol red in photo-experiments) with glutamine and penicillin/streptomycin.

Instruments
NMR spectra were recorded on a Bruker AV-400/300 spectrometer. Elemental analysis was performed by Exeter Analytical using a CHN/O/S Elemental Analyser (CE440). Positive ion ESI-MS spectra were obtained using an Agilent 6130B single quad coupled to an automated sample delivery system (isocratic Agilent 1100 HPLC without column) . UV-visible absorption spectra were recorded in 1-cm cuvettes on a Varian Cary 300 UV-vis spectrophotometer. The fluorescence spectra were recorded on a JASCO FP-6500 Fluorimeter (band width (ex): 5 nm; band width (em): 5 nm; ex = 405 nm; em = 450-650 nm). The EPR measurements were carried out at ambient temperature on a Bruker EMX spectrometer. The IR spectra were recorded on a PerkinElmer Fourier Transform Infrared Spectrometer. The confocal images were visualized using Zeiss 710 or 880 confocal microscopy (63 oil-immersion objective).

Synthesis of the Maleimide Ligand (E)
Overall synthetic scheme:
The mixture was stirred for 10 h at room temperature. After the reaction, solvents were reduced to 3 mL, NH4PF6 was added, the yellow solid was filtered and washed with water and diethyl Then, the absorbance of the generated 2-TP was determined and plotted against the concentration of the reagent.

Synthesis of Ir1-HSA Conjugate
The iridium complex (0.4 mmol) was dissolved in 20 mL methanol and water (v/v 1:2). Then HSA (0.4 mmol) in water was added. The mixture was stirred for 1 h and purified by dialysis (10 kDa MWCO membrane). The purified Ir1-HSA conjugate was collected and freeze dried. The concentration of Ir was determined by Inductively-Coupled-Plasma Optical Emission S5 Spectroscopy (ICP-OES).

Electron Paramagnetic Resonance (EPR) Experiment
An LED lamp (465 nm) was used as light source. The sample was contained in a flat-cell The samples were digested overnight in 60% HNO3 at ambient temperature for 24 h. Each sample was then diluted with Milli-Q water to give a 3% HNO3 sample solution. The iridium content was determined by inductively-coupled-plasma mass spectrometry (ICP-MS, Agilent 7500 series).

Detection of Intracellular 1 O2
Intracellular ROS generation by Ir1 or Ir1-HSA after irradiation was detected using a red fluorescence cellular reactive oxygen species detection kit (ab186027, abcam). Firstly, the cultured cancer cells (10 5 cells) were incubated with 100 L/well of the ROS probe working solution for 1 h in the incubator, then treated with Ir1 or Ir1-HSA in the dark for 2 h, and washed with PBS. The 'light' plate was subjected to blue light irradiation for 5 min. The intensity of the red fluorescence was measured on a microplate reader (Promega) immediately. The excitation wavelength of the ROS probe was 520 nm, and the fluorescence was collected at 590-625 nm.  Figure S1. UV-vis absorption spectra of Ir1 in PBS (containing 2% DMSO) (a) before and after 12 h in the dark, and (b) before and after blue light irradiation (465 nm) for 1 h.