Centrifugation filters

Harvesting and culturing of primary cardiac fibroblasts were conducted as previously described ^{27 (link)}. For immunoblotting, cells were grown to 80–90% confluence. Cells were then washed three times with PBS to remove residual FBS before being serum starved in DMEM supplemented with 10 μg/ml TGFβ (R&D), 100 μg/ml L-ascorbic acid (Sigma), and 40 μg/ml soybean trypsin inhibitor (Sigma). Twenty-four hours post starvation; conditioned media were collected, and concentrated by centrifugation filters (Millipore).

To quantitatively determine the release of doxorubicin, AuNPs B were suspended in phosphate buffer (pH 7.4) or acetate buffer (pH 5.0) at 1.5 mg mL⁻¹ and stirred for 30 h. The fluorescence emission spectra were recorded every 2 h. Next the nanoparticle solution was centrifuged (Milipore, centrifugation filters) to remove supernatant that contained released doxorubicin. This was further lyophilized and the amount of doxorubicin in given nanoparticles preparation was quantified using HPLC with solvent gradient from 20 to 50% (0.01M TFA:acetonitrile) for 25 min/injection. The amount of doxorubicin was in agreement with the fluorescence intensity measurements.