Polymer encapsulation of anticancer silver–N-heterocyclic carbene complexes

Amphiphilic block copolymers have been developed for the encapsulation of organometallic drugs. silver–N-heterocyclic carbene complexes have shown significant promise as anticancer and antibacterial compounds, and have been studied as the payload in these carriers. Simple modification of the N-heterocyclic carbene ligand structure enables solubility properties and interaction with the polymer to be tuned.


General
All chemicals were purchased from Sigma-Aldrich, Alfa Aesar or Thermo Fisher Scientific and used as supplied.
1 H and 13 C{ 1 H} NMR spectra were recorded on either a Bruker DPX300 spectrometer (operating frequency 300.1 MHz for 1 H and 75.48 MHz for 13 C{ 1 H}), a Bruker Avance 500 spectrometer or a Bruker DRX500 spectrometer (both with an operating frequency of 500.13MHz for 1 H and 125.80 MHz for 13 C{ 1 H}).All spectra were recorded at 298K in deuterated solvent.Chemical shift values are quoted in parts per million (ppm, δ), coupling constants, J, are quoted in Hertz (Hz) and assignment of 13 C{ 1 H} NMR spectra was aided by 13 C{ 1 H} DEPT135 experiments when necessary.
High-resolution mass spectra were collected on a Bruker Daltonics (micro TOF) instrument operating in the positive ion electrospray mode.Samples were injected directly from feed solutions and acquired over the range m/z 50 -4000.All spectra were recorded using an acetonitrile/water mix as the eluent and a sodium formate solution as a calibrant.
Microanalyses were performed in the School of Chemistry, University of Leeds by Ms. Tanya Marinko-Covell using a Carlo Erba Elemental Analyser MOD 1106 spectrometer.
FTIR spectra were recorded using a Spectrum One spectrophotometer (PerkinElmer) fitted with diffuse reflectance probe with zinc-selenide window.IR spectra were recorded using a globular light source through KBr beamsplitter for the range 4000-400 cm -1 using DLATGS detector with KBr window.32 scans were recorded for each averaged spectrum with a new background recorded after each sample.IR spectra were analysed using the spectroscopy software package OPUS (v 6.5, Bruker Optiks GmbH).
Scanning Electron Microscopy (SEM) experiments were performed using a Pasteur pipette to extract a drop of the solution and deposit it on to an SEM glass cover slip.
The sample was allowed to dry in a fume cupboard, after which the cover slip was mounted on an SEM stub using conductive tape.The SEM stub containing the dried sample was sputter-coated with a thin layer of gold using a current of 20 mA for 2 minutes, in a quorum Q150RS sputter-coater.The coated samples were analysed for particle size and morphology using a JOEL JSM-6610LV microscope (Oxford Instruments) equipped with a field emission electron gun as an electron source, using a working distance of 11 mm.Accelerated voltage was applied between 5 and 15 kV.Fluorescence imaging of was performed using a confocal microscope Zeiss LSM88o inverted with Airyscan microscope that consists of Axio Observer Z1, Nano Focusing Piezo Stage and Zlmulti S1 incubator box.The images were taken at 63 x objective.
Complex C4: L4 (0.2 g, 0.42 mmol) and silver oxide (0.19 g, 0.84 mmol) were added to activated molecular sieves 4Å in a Schlenk flask and dried in vacuo.Anhydrous MeCN (10 mL) and anhydrous MeOH (10 mL) were transferred to the Schlenk flask and the mixture was stirred at room temperature for 24 hours.The solution, which had turned grey, was filtered through celite, and the solvent removed from the filtrate in vacuo to yield the product as a white solid.Yield: 0.15 g, 0.2 mmol, 46 %. 1

Synthesis of Cbz-protected P2-P4
A representative procedure is given for a target monomer-initiator molar feed ratio of 40 (P3).Lys(Cbz) NCA (380.2 mg, 1.24 mmol) was dissolved in anhydrous DCM (10 mL).The solution was injected into a flame-dried and nitrogen-purged Schlenk tube.Amine-terminated mPEG113, average Mn = 5,000 g/mol (155 mg, 0.031 mmol) was dissolved in anhydrous DCM (10 mL).The solution was injected into the reaction medium.Further steps were carried out as described for the synthesis of

Deprotection of Cbz-protected P2-P4
Protected polymer (480 mg) was dissolved in TFA (7 mL) and added to a 33 wt.% solution of HBr in TFA (3 mL).The mixture was stirred at room temperature for 24 hours, then added dropwise into cold Et2O (150 mL) to induce the precipitation of the deprotected polymer.The precipitate was isolated by centrifugation (3000 rpm, 10 min).The polymer was re-suspended in fresh Et2O, stirred for 30 min and centrifuged.This was repeated several times until a clear supernatant was obtained.
The polymer was dissolved in de-ionised H2O and dialysed against de-ionised water for 96 hours, with the dialysate being substituted with a fresh supply after every 8 hours.The polymer was isolated by freeze-drying.

Creation of nanoparticles by silver-NHC complex-mediated polymer selfassembly
Solutions (1 mg/mL) of P2, P3, P4, L3, L4, C3 and C4 were prepared in HPLC grade water, and filtered through 0.45 µm and 0.2 µm filters and analysed using DLS to confirm no particles were present in the absence of polymer-silver-NHC complex interaction.Equal volumes (1 mL) of each polymer solution and either ligand or complex were mixed to form solutions of 1:1 ratio, and agitated using a vibrax machine.The particles in solution were maintained under dialysis to determine any non-uptake of inorganic matter, heated to 37 °C and analysed by DLS (Table 1  hours at 37 ºC in an atmosphere of 5 % CO2 prior to drug exposure.All compounds were dissolved in DMSO at a concentration of 100 mM and diluted with medium to obtain drug solutions ranging from 100 µM to 0.049 µM.The final DMSO concentration was 0.1 % (v/v) which is non-toxic to cells.Drug solutions were applied to cells and incubated for 96 hours at 37 ºC in an atmosphere of 5 % CO2.The solutions were removed from the wells and fresh medium added to each well along with 20 µL MTT (5 mg/mL), and incubated for 4 hours at 37 ºC in an atmosphere of 5 % CO2.The solutions were removed and 150 µL DMSO was added to each well to dissolve the purple formazan crystals.A plate reader was used to measure the absorbance at 540 nm.Lanes containing medium only, and cells in medium only (no drug), were used as blanks for the spectrophotometer and 100 % cell survival respectively.Cell survival was determined as the absorbance of treated cells divided by the absorbance of controls and expressed as a percentage.The concentration required to kill 50 % of cells (IC50) was determined from plots of percent survival against drug concentration.Each experiment was repeated 3 times and a mean value obtained.