Methyl methacrylate, acrylic acid, and poly(ethylene glycol) (n) diacrylate (n = 200 = MW of PEG block) were purchased from Polysciences, Inc. (Warrington, PA, USA) and used as received. Potassium persulfate, copper sulfate, copper acetate, nitric acid (trace metal grade) were from Fisher Scientific (Pennsylvania, PA, USA). All materials were used as received unless otherwise noted. Microwave reactions were conducted in a Synthos 3000 from Anton Paar (Ashland, VA, USA). ICP MS experiments were conducted on a Varian 820-MS (Varian Inc., Lake Forest, CA, USA) using the following parameters: plasma flow 17.5 L/min, auxiliary flow 1.65 L/min, sheath gas 0.13 L/min, and nebulizer flow 0.89 L/min. The torch alignment had a sampling depth of 5 mm. The RF power was set at 1.3 kW. The pump rate was 3 rpm, and the stabilization delay was 30 s. The ion optics parameters were: first extraction lens -1 V, second extraction lens -191 V, third extraction lens -206 V, corner lens -236 V, mirror lens left 56 V, mirror lens right 49 V, mirror lens bottom 16 V, entrance lens 0 V, fringe bias -2.5 V, entrance plate -31 V, and pole bias 0 V. CRI parameters were skimmer gas off, sampler gas off, skimmer flow 0 mL/min, and sampler flow 0 mL/min. ICP MS tubing was rinsed in between samples to avoid sample contamination.
Synthesis of nanoparticles
An aqueous solution (58.8 mL) containing acrylic acid (0.57 g), methyl methacrylate (0.575 g), PEG diacrylate (0.053 g), and potassium persulfate (0.164 g) was prepared in a PTFE vessel for a Synthos 3000 16MF100 rotor in a freshly regenerated inert atmosphere glovebox. The vessel was sealed, removed from the glovebox, and placed in the 16MF100 rotor along with seven other vessels containing 60 mL of water each. The rotor was placed in the microwave and then heated to 90°C for 60 min with a maximum microwave power of 1400 W (see Additional file 5). The internal temperature and pressure of the vessel containing the monomer solution were monitored via a p/T sensor accessory (Anton Paar). The resulting nanoparticle solution was dialyzed in 4 L of ultrapure water for 48 h with a change in the water after the first 24 h. The particle concentration after purification was determined by lyophilizing a known volume and then weighing the resulting solid, which resulted in a final particle concentration of 12.8 mg/mL. Based on this number, a total of 0.896 g of particles was synthesized with approximately 75% conversion of monomer to particles.
A 3-mL aliquot of the nanoparticle solution was adjusted to a pH of 7 using NaOH followed by the addition of copper sulfate in a 1:1 molar ratio with amount of NaOH added. The particle solution was then dialyzed in 1.5 L of ultrapure water for 48 h to remove unbound copper. Particle size of approximately 215 nm was determined via dynamic light scattering (DLS).
ICP MS Cu-loading studies
For Cu-loading studies, the Cu-loading solution containing CuSO4 and nanoparticles was dialyzed in 1.5 L of ultrapure water. Samples (1 mL each) were removed at 1, 2, 3, 4, 5, 6, 12, 24, and 48 h, diluted in 1% nitric acid and then analyzed for63Cu content via ICP MS. Cu content was determined by comparison with a calibration curve generated using known samples (see Additional file 6).
ICP MS Cu release studies
Purified CuCNP-containing solutions (3 mL) were dialyzed in 1.5 L of the desired buffering solution. Samples (1 mL each) were removed at 0.08, 1, 2, 3, 4, 5, 6, 12, 24, and 48 h, diluted in 1% nitric acid and then analyzed for63Cu content via ICP MS. Cu content was determined by comparison with a calibration curve generated using known samples.
X-ray photoelectron spectroscopy
XPS spectra were acquired with a PHI 5000 VersaProbe™ Scanning XPS Microprobe (Physical Electronics Inc., Chanhassen, MN, USA). Samples were prepared by spotting 5 μL of the desired particle-containing solution onto a glass slide and then drying under vacuum.
Scanning electron microscopy and energy-dispersive X-ray analysis
SEM images and EDX spectra were obtained with a Quanta ESEM microscope (FEI, Hillsboro, OR, USA) equipped with a Sapphire Si(Li) detecting unit for EDX (EDAX Inc., Mahwah, NJ, USA). Samples were prepared by spotting 5 μL of the desired particle-containing solution onto a glass slide, drying under vacuum, and then repeating the spot/dry three times to produce samples with enough thickness to prevent interference from the glass slide during EDX analysis. Samples were then coated with Au (2-5 nm thickness) using a Cressington 108 Manual Sputter Coater (Ted Pella, Redding, CA, USA). Images were obtained with an acceleration voltage of 5-15 kV and EDX spectra were obtained with an acceleration voltage of 5 kV.
Microanalysis was performed by Columbia Analytics (formerly Desert Analytics) in Tucson, AZ. Samples (100 mg) for elemental analysis were prepared by lyophilizing the desired nanoparticle-containing solution, which were further dried for 4 h at 25°C under vacuum prior to analysis.
Cell viability measurements
HeLa cells were purchased from ATCC (cat. # CCL-2), and maintained in Eagle's Minimum Essential Medium (ATCC, cat. # 30-2003) with 10% FBS (Thermo Scientific HyClone, South Logan, UT, USA). Five thousand cells per well seeded on 96-well plates and incubated overnight at 37°C (5% CO2). The desired particle amounts were added to the wells and the plates were incubated for an additional 48 h at 37°C (5% CO2). After the incubation, cell viability was evaluated with the MTT reagent. Media was removed each well and replaced with fresh media containing 1 mg/mL MTT. The cells were incubated for 4 h at 37°C (5% CO2) after which time the media was removed and replaced with DMSO. Light absorption was measured on a Synergy 2 multi-mode microplate reader (BioTek, Winooski, VT, USA). The viability of the cells exposed to particles was expressed as a percentage of the viability of cells grown in the absence of particles on the same plate.