In vivo metallophilic self-assembly of a light-activated anticancer drug


 Self-assembling molecular drugs combine the easy preparation typical of small-molecule chemotherapy and the tumor-targeting properties of drug-nanoparticle conjugates. However, they require a supramolecular interaction that survives the complex environment of a living animal. Here, we report that the metallophilic interaction between cyclometalated palladium complexes generates supramolecular nanostructures in living mice that have a long circulation time (above 12 h) and efficient tumor accumulation rate (up to 10.2% ID/g) in a skin melanoma tumor model. Green light activation leads to efficient tumor destruction due to the photodynamic effect generated by the self-assembled palladium complexes, as demonstrated in vitro by an up to >96-fold cytotoxicity increase upon irradiation. This work demonstrates that metallophilic interactions are well suited for generating stable supramolecular nanotherapeutics in vivo with exceptional tumor-targeting properties.


Materials and methods
All reagents were purchased from commercial vendors. The reactants and solvents were used without further purification. All 1 H NMR, 13 C attached-proton-test NMR ( 13 C-APT NMR) were obtained on a Bruker DPX-300 spectrometers. Chemical shifts are indicated in ppm relative to the residual solvent peak. Electrospray ionization mass spectra (ESI-MS) were recorded by using an MSQ Plus Spectrometer positive ionization mode. The TEM experiments were carried via TEM JEOL 1010: 100 kV transmission electron microscope using Formvar/Carbon coated copper grid from Polysciences Inc. Uv-vis spectra were recorded on a Cary 50 spectrometer from Varian. The emission spectra and relative phosphorescence quantum yields were measured via an FLS900 Spectrometer from Edinburgh Instruments Ltd. The phosphorescence lifetime of the complexes in water was measured on a LifeSpec-II spectrometer from Edinburgh Instruments, using as excitation source a 375 nm pulsed diode laser. The singlet oxygen emission spectra were measured on a special custom-built setup which was described previously.(1) The DFT calculations were carried out using the Amsterdam Density Functional software (ADF2019) from SCM, the PBE0 functional, a triple zeta basis set (TZP), and COSMO to simulate the solvent effect in the water. Human cancer cell lines A549 (lung carcinoma), A431 (skin carcinoma) and A375 (malignant melanoma) were distributed by the European Collection of Cell Cultures (ECACC) and purchased from Sigma Aldrich.
Dulbecco's Modified Eagle Medium (DMEM, with and without phenol red, without glutamine), Glutamine-S (GM; 200 mm), tris(hydroxylmethyl)aminomethane (Tris base), trichloroacetic acid (TCA), glacial acetic acid, and sulforhodamine B (SRB) were purchased from Sigma Aldrich. Opti-MEM Reduced Serum Media without phenol red was obtained from Gibco. The measurements of complexes on photocytotoxicity were performed according to the literature. (2) Annexin V/propidium iodide double staining assay was purchased from Bio-Connect BV. The FractionPREPTM Cell Fractionation kit was obtained from BioVision Incorporated.
Cryo-EM images were collected on a Titan/Krios operating at 300 kV at a nominal magnification of 33000x or 81000x yielding a pixel size at the specimen of 3.5Å or 1.4 Å, respectively (NeCEN, Leiden University).

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2D-monolayer photocytotoxicity experiments. For the cytotoxicity assay, Opti-MEM complete medium without phenol red was used, supplemented with 2.5% v/v fetal calf serum (FCS), 0.2% v/v penicillin/streptomycin (P/S), and 1% v/v Glutamine). Briefly, 100 µL Opti-MEM complete medium suspensions of A549 (5000 cells), A431 (8000 cells), or A375 (5000 cells) cells, were seeded into 96-wells plates and separated as dark or light groups, and incubated in the normoxic (21% O2, 37 °C) or hypoxic (1% O2, 37 °C) incubators. After 24 h, the cells were treated with PdL (100 µL) in a series of concentrations. At 48 h, the cell plates in the light group were irradiated with 520 nm green light with a dose of 13 J/cm 2 (normoxic-2D: 20 min, 10.92 mW/cm 2 ; hypoxic-2D: 32 min, 6.90 mW/cm 2 ), in normoxic (21% O2) or hypoxic (1% O2) conditions, while the dark group was kept in the dark. After irradiation, the cells were incubated in the dark for another 48 h. Then 100 µL of TCA fixation solutions (10% w/v) were added to the wells, and the plates were kept at 4 °C for 24 h. The photocytotoxicity of the complex was determined via the sulforhodamine (SRB) assay, and the normoxic or hypoxic half-maximal effective concentrations EC50 were obtained via Graphpad 8 using the dose-response two-parameter Hill-slope equation 1. Data are averages (n=3) with 95% confidence intervals (in µM) over three independent experiments.
3D tumor spheroids viability assay. 100 µL Opti-MEM complete medium suspensions of A549 (500 cells), A431 (500 cells), or A375 (300 cells in normoxic conditions, 1000 cells in hypoxic conditions) cells were seeded into 96-well round-bottom Corning spheroid microplates and split as dark or light groups. Each plate was incubated for 3 days in normoxic or hypoxic conditions, to obtain 3D tumor spheroids. Then, the spheroids were treated with PdL (100 μL Opti-MEM complete medium) in a concentration series (0, 0.05, 0.25, 0.5, 1, 1.25, 2.5, 5, 12.5, 25). 24 h later, the plates of the light group were irradiated with 520 nm green light with a dose of 13 J/cm 2 (normoxia-3D spheroid condition: 32 min, 6.90 mW/cm 2 ; hypoxia-3D spheroid condition: 55 min, 3.99 mW/cm 2 ) and incubated for another 48 h. Then a CellTiter Glo 3D solution (50 μL/well) was added to each well to stain the 3D tumor spheroids. After 30 min shake on an IKA Vibrax shake at 500 rpm at room temperature, the luminescence in each well was measured by a Tecan Microplate Reader. Half-maximal effective concentrations (EC50) for 3D tumor spheroids growth inhibition were calculated by fitting the CellTiter Glo3D doseresponse curves using the same non-linear regression function as in 2D (Equation 1) as implemented in Graphpad Prism 8. Data are averages (n=3) with 95% confidence intervals (in µM) over three independent experiments.

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Mode of cell death study using flow cytometry. 2×10 5 A375 cells were seeded in 12-well plates that were separated into dark and light groups, and incubated in normoxic condition.
After 24 h, the cells were treated with PdL at a final concentration at 0.5 µM or 2 µM, and incubated for 24 h. Then, the light groups were irradiated with 520 nm green light with a dose of 13 J/cm 2 (20 min, 10.92 mW/cm 2 ), and incubated in normoxic condition for 2 h, 4 h or 24 h.
Afterward, the cells in all groups were harvested with trypsin and stained with Annexin propidium iodide (535/617 nm). All flow cytometry data were processed using FlowJo10.

Cellular uptake experiments.
To measure simple cellular uptake, A375 cells (2×10 5 ) were seeded in 12-well plates and incubated for 48 h. Then the cells were treated with PdL (2 µM, 1 mL) for 2 h or 24 h. After that, the cells were washed by PBS for one time, and then harvested and centrifuged. After removing the supernatant, 0.5 mL of 65% HNO3 was added to lyse the cell pellets with an overnight shake. For cellular uptake inhibition experiments, the cells were pretreated with different inhibitors for 1 h (NaN3 (1 mg/mL), pitstop 2 (20 µM), dynasore (80 µM), nocodazole (40 µM), and wortmannin (4 µM)), or incubated at 4 °C for 30 min. Then, the cells were treated with PdL (5 µM) and incubated either in normoxic conditions (37 °C, 5% CO2, 21% O2, 100% humidity) or at 4 °C (in the air condition) for another 2 h. After that, the cells were harvested, centrifuged and lysed using the same method as in absence of inhibitor.
Then, 9.5 mL of milli-Q water was added to the cell lysis solution to lower the HNO3 concentration to 3.25% (v/v). The Pd content in the solution samples was measured via ICP-

In vivo tumor inhibition experiments.
Female BALB/c mice with 3 weeks old were originally purchased from Vital River Laboratory Animal Center (Beijing, China). The mice were kept under specific pathogen-free conditions with free access to standard food and water for 2 weeks, to let the mice weight around 20 g. This study was conducted following the Guide for the Care to obtain mouse A375 melanoma implant. 3 weeks later, the tumor volumes were around 100 mm 3 . Tumor volume (V) can be calculated by formula V = L/2 × W 2 after measuring the tumor length (L) and width (W). (3) The mice were then randomly divided into 4 groups (vehicle control, 520 nm light, PdL, PdL + 520 nm light groups, each group 4 mice). The injectable PdL solution were prepared by diluting the PdL stock DMSO solution (4.2 µM) to 420 µM using DMEM medium containing 10% v/v fetal bovine serum (FBS) and 1% v/v penicillin/ streptomycin (P/S). The mice were treated through tail intravenous injection with DMEM for vehicle control and 520 nm light groups, or PdL (2.1 µmol/kg, 420 µM, 100 µL DMEM medium (10% FBS), 0.9 mg/kg) for PdL dark and PdL + 520 nm light groups. After 12 h injection, 520 nm irradiation (100 mW/cm 2 , 5 min) was then carried out twice, with an interval of 5 min for the light groups. Thus, the total light dose for each treatment was 100 mW/cm 2 , 10 min, 60 J/cm 2 . These treatment and irradiation steps were replicated at day 0, day 7 and day 14, respectively. On day 5, one mouse in each group was sacrificed and the tumor were taken up and fixed with paraformaldehyde (10 % v/v), then sectioned into slices and analyzed via H&E or TUNEL protocols, to evaluate the tumor cell damage and apoptosis conditions. The tumor volume and body weight of left mice (N=3) were measured and recorded and the average tumor volume and body weight were calculated over 20 days. At last, the mice were sacrificed, and the healthy organs were taken up, fixed with paraformaldehyde (10% v/v), then sectioned into slices and analyzed via H&E protocol, to determine their side effect after treatment.

Mice blood EM imaging experiments.
The tumor-bearing mouse was treated with PdL (2.1 µmol/kg, 420 µM, 100 µL DMEM medium (10% FBS), 0.9 mg/kg) through intravenous tail injection. After 5 min, 1 mL of blood was taken up from the eye socket and diluted to 5 mL by PBS. After centrifugation (1500 rpm, 10 min), the supernatant was collected, and the left part was washed by PBS (5 mL) and centrifuged (1500 rpm, 10 min) again twice more, to obtain the supernatant PBS solution. These PBS solutions were then combined and centrifuged at a speed of 10000 rpm for 10 min.