Synthesis and Biological Evaluation of Homogeneous Thiol‐Linked NHC*‐Au‐Albumin and ‐Trastuzumab Bioconjugates

Abstract Targeted delivery of potent cytotoxic drugs to cancer cells minimizes systemic toxicity and several side effects. NHC*−Au−Cl has already been proven to be a potent anticancer agent. In this study, we explore a strategy based on chemoselective cysteine conjugation of NHC*−Au−Cl to albumin and trastuzumab (Thiomab LC‐V205C) to potentiate drug‐ligand ratio, pharmacokinetics, as well as drug efficacy and safety. This strategy is a step forward towards the use of gold‐based anticancer agents as targeted therapies.


General procedures for protein/antibody labelling and characterization of the bioconjugates General procedure for protein and antibody conjugation with NHC*-Au-Cl
To an eppendorf tube with NaPi (50 mM, pH 7.0) and DMF (10% of total volume), an aliquot of a stock solution of protein (final concentration 10 µM) was added. Afterwards, a solution of the NHC*-Au-Cl (1 to 10 equiv.) in DMF was added and the resulting mixture was vortexed for 10 seconds. The reaction was mixed for 2 or 24 h, at 37 ºC. A 10 µL aliquot of each reaction time was analysed by LC-MS and conversion to the expected product was observed.

LC-MS method for analysis of protein conjugation
LC-MS was performed on a Water Acquiry UPLC system equipped with a single quadrupole mass detector using an Acquity UPLC protein BEH C4 column, 300 Å, (1.7 mm, 2.1 × 50 mm). Solvents A, water with 0.01% formic acid and B, 71% acetonitrile, 29% water and 0.075% formic acid were used as the mobile phase at a flow rate of 0.2 mL·min -1 from 0-20 min, and 0.05 mL·min -1 from 20-30 min. The electrospray source was operated with a capillary voltage of 3.0 kV and a cone voltage of 20 V. Nitrogen was used as the desolvation gas at a total flow of 8 L·h -1 . Total mass spectra were reconstructed from the ion series using the MaxEnt algorithm preinstalled on MassLynx software (v. 4.1 from Waters) according to the manufacturer's instructions. To obtain the ion series described, the major peak(s) of the chromatogram were selected for integration and further analysis.
UPLC for protein/antibody analysis: deionized water containing 0.1% of formic acid (solvent A) and 71% MeCN, 29% H2O, 0.075% formic acid (solvent B) are used as mobile phase for gradient elution. The gradient program for the purification of protein/antibody is reported in the following table. *(flow 0.2 mL/min from 0-20 min, flow 0.05 mL/min from 20-30 min).

Analysis of protein conjugation by LC-MS
A typical analysis of a conjugation reaction by LC-MS is described below. Briefly, the total ion chromatogram, combined ion series and deconvoluted spectra are measured for the staring material (rHSA). This allows to monitor progress of the conversion of the nonmodified protein/antibody to the conjugated protein/antibody. After size exclusion purification, Bradford protein analysis is used to determine the yield of the reaction.
Identical analyses were carried out for all the conjugation reactions performed in this work.
-4 -Supporting Figure 1. A typical analysis of a conjugation reaction by LC-MS is described for the rHSA. The total ion chromatogram, combined ion series and deconvoluted spectra are shown. Identical analyses were carried out for all the conjugation reactions performed in this work.

Stability of bioconjugates in human plasma
A 20 µL aliquot of the NHC*-Au-rHSA (10 µM) in NaPi buffer (50 mM, pH 7.0) was thawed. 1 µL of reconstituted human plasma was added at room temperature and the resulting mixture vortexed for 10 seconds. The resulting reaction mixture was then mixed at 37 ºC. After 24 and 48 h, a 10 µL aliquot of each reaction mixture was analysed by LC-MS.
Afterwards, a solution of NHC*-Au-Cl (1, 5, 10 or 50 equiv.) in DMF was added and the resulting mixture was vortexed for 10 seconds. The reaction was mixed for 2 h at 37 ºC.
Small molecules were removed from the reaction mixture by loading the sample onto a Zeba Spin Desalting Column previously equilibrated with NaPi (50 mM, pH 7.0).* The sample was eluted via centrifugation (2 min, 1500xg). A 10 µL aliquot was analysed by LC-MS and full conversion to the expected product was observed (calculated mass, 67037 Da; observed mass, 67037 Da). * These columns are described to have at least 95% retention (removal) of salts and other small molecules (<1000 MW). However, when the reaction was scaled up for in vitro studies, this procedure was followed by a dialyses to optimize the efficiency of the method. The sample was dialysed against 0.5 L of NaPi (50 mM, pH 7.0), stirring, overnight, at room temperature. The buffer solution was changed after 2 h. The following day, the buffer solution was changed once again. After 24 h, the sample was analysed by LC-MS and the concentration determined with a SpectraMax i3x.

Viability Assays
Cells were seeded at 10,000 cells per well into flat-bottom 96-well plate. NHC*-Au-Cl and NHC*-Au-Thiomab were added after allowing the cells to settle overnight. Relative viability was assessed by using the CellTiter Blue assay (Promega) according to manufacturer's instructions. Fluorescence was determined after 24 h of incubation at 37 °C on a fluorescent plate reader (TECAN Infinite M200 -excitation/emission 560/590 nm) and obtained values were normalized to the respective controls.