Nanoparticle surface charge mediates the cellular receptors used by protein-nanoparticle complexes.

Nanoparticles (NPs) Fluorescent polystyrene NPs (FluoSpheres, Invitrogen) were used in all cellular binding experiments. The diameter provided by the supplier is used to denote the NP: 87 nm amine-modified ( C29029 ), 200 nm amine-modified (F8764), 40 nm carboxylate-modified (F8795), and 200 nm carboxylate-modified (F8811) NPs. Experimental values are provided in Table 1 . Dynamic Light Scattering (DLS) and Zeta Potential Measurements The hydrodynamic diameter and zeta potentials of the NPs were measured with a Malvern Zetasizer (Nano-ZS, Malvern Instruments) in disposable cuvettes. Solutions of NPs in water were measured at the following concentrations: 87 nm amine-modified (173 pM); 200 nm amine-modified (15 pM); 200 nm carboxylate-modified (13 pM); and 40 nm carboxylate-modified (3.3 nM). Measurements were carried out in triplicate. Hydrodynamic diameter and zeta potential data was acquired from ≥12 runs per measurement and 30 runs per measurement, respectively. Zeta potential measurements were run in general purpose mode and the Smoluchowski approximation was used to convert the electrophoretic mobility to a zeta potential.

Cell Culture

African green monkey kidney epithelial cells (BS-C-1, ATCC) were maintained in a 37 °C, 5% carbon dioxide environment in Minimum Essential Medium (MEM, Invitrogen, 61100061) with 10% (v/v) fetal bovine serum (FBS, Invitrogen, 10437028). Cells were passaged every 3 days. For fluorescence imaging, cells were cultured in 35 mm glass-bottom cell culture dishes (MatTek). Nuclei were stained with 27 μM 4′,6-diamidino-2-phenylindole dilactate (DAPI, Invitrogen, D3571) at 37 °C for 1 hour in MEM supplemented with 10% FBS.

Fluorescence Microscopy

The cellular binding of NPs was imaged with an epifluorescence microscope (Olympus IX7) using a 1.20 N.A., 60x, water immersion objective (Olympus). Emission was detected with an EMCCD (DU-897, Andor). All images for comparison were acquired with the same exposure time and gain. Image J ( http://rsb.info.nih.gov/ij/ ) was used for analysis. Brightness and contrast were set equally for all images for comparison.

Gel Electrophoresis

NPs incubated in MEM supplemented with 10% FBS were washed by repeated centrifugation (16,000 × g for 10 minutes) and resuspension in water. The supernatant was collected after each wash. After the final wash, the sample was suspended in buffer containing 6% SDS (New England Biolabs, #B7703S) to remove the protein from the NP surface. Supernatant was diluted by 50% in Laemmli buffer (Boston Bioproducts, BP-110R), boiled for 5 min, and then loaded onto the gel. The supernatant from the first wash was diluted by an additional factor of 10 in water to avoid overloading the gel. The undiluted supernatant is shown in Figure S1 . Proteins were separated on a 4–20% gradient mini-protean gel (Bio-Rad, 456–1094) at 40 mA and 130 V along with a 5–225 kDa molecular weight marker (Lonza, 50547). Proteins were stained with Simply Blue Safe Stain (Invitrogen, LC6060) for one hour.

Competition Assay

Binding competition studies used polyinosinic acid (Sigma-Aldrich, P4154) as a competitor for scavenger receptors and polyadenylic acid (Sigma-Aldrich, P9403) as a control. The competitor or control was incubated with the cells for 20 minutes in MEM supplemented with 10% FBS prior to the addition of amine-modified NPs or MEM alone prior to the addition of carboxylate-modified NPs. Cells were incubated with NPs at 4 °C in the presence of competitor for 10 minutes. Cells were rinsed twice with PBS with calcium and magnesium (Invitrogen, 14040182) and twice with PBS without calcium and magnesium (Invitrogen, 14190250). To remove the cells from the MatTek dishes and put them into suspension for flow cytometry (BD LSR II, BD Biosciences), the cells were incubated in a 10 mM solution of ethylenediaminetetraacetic acid (EDTA, Mallinckrodt, 4931-04) in PBS without calcium or magnesium for 30 minutes at 37 °C. Cells were rinsed twice with Leibovitz’s L-15 buffer (Invitrogen, 21083027) by centrifugation at 10,000 × g for 8 minutes or 5,000 × g for 5 minutes (200 nm carboxylate-modified NPs) and kept on ice for at least one hour prior to flow cytometry. NP fluorescence was excited with a 488 nm excitation source and collected with a 530/30 nm bandpass filter. Between 5,000–14,000 cells were used to calculate the mean fluorescence (n = 3–5). Histograms were analyzed using Weasel 3.0.1 (Walter and Eliza Hall Institute of Medical Research, Victoria, Australia).

Supplementary Material 1_si_001