Oligonucleotide Loading Determines Cellular Uptake of DNA- Modified Gold Nanoparticles

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1 Supporting Information for: Oligonucleotide Loading Determines Cellular Uptake of DNA- Modified Gold Nanoparticles David A. Giljohann, Dwight S. Seferos, Pinal C. Patel, Jill E. Millstone, Nathaniel L. Rosi, and Chad A. Mirkin* Materials and Methods DNA Nanoparticle (ASNP) preparation Citrate stabilized gold nanoparticles (Au NP) (13 ± 1nm) were prepared using published procedures 1. Thiolated oligonucleotides, 28 bases in length (ODNs) consisting of a block of 18 mixed bases, a block of 10 adenine bases, and a 3 -thiol modifier (5 -GAG CTG CAC GCT GCC GTC AAA AAA AAA A- SH- 3 ), were synthesized on an Expedite 8909 Nucleotide Synthesis System (ABI) using standard solid-phase synthesis and reagents (Glen Research). All sequences were HPLC purified following synthesis. The ASNPs were prepared using previously published methods 2. Briefly, thiol-modifed oligonucleotides (3 μm) were added to Au NPs (10 nm) in Nanopure water (18.2 MΩ). The solution was brought to concentrations of 0.01% SDS, 0.01 M phosphate buffer ph 7.4, and 0.1M NaCl. The solution was further aged with additions of NaCl over 12 hrs to bring the final NaCl concentration to 0.3M. Mixed monolayer particles containing oligo (ethylene glycol) (OEG) and DNA were synthesized by incubating Au NPs (10 nm) in a aqueous mixture of thiolated oligonucleotides (1.5 μm) and (HO-(CH 2 CH 2 O) 3 -C 11 H 22 - SH) (OEG). The stoichiometry of OEG was varied from 0.2 to 50 (relative to thiolated DNA) to control the loading of the oligonucleotides on the particles. Functionalized nanoparticles were separated from free oligonucleotides via three consecutive centrifugation steps (13,000 rpm, 20 min) and washed with phosphate buffer saline solution (PBS) (137 mm NaCl, 10 mm phosphate, 2.7 mm KCl, ph 7.4, Hyclone) after each centrifugation interval. Finally, the particles were re-

2 suspended in PBS buffer and filter sterilized using a 0.2 µm acetate syringe filter (GE). Particle concentrations were determined by measuring extinction at 524 nm on a UV/visible spectrophotometer (Agilent Technologies). The particle DNA loading was determined using a modification of literature procedures 3. Instead of covalently labeling the oligonucleotides with a flourophore, we measured their concentration with an OliGreen ssdna quantification assay (Invitrogen) upon oxidative dissolution of Au with KCN. Cell Culture Models The uptake of ASNP agents was studied using several model cell lines. C166 (mouse endothelial), HeLa (human cervical carcinoma), and A549 (human lung carcinoma) were obtained from ATCC. C166, HeLa, and A549 cells were maintained in Dulbecco s modified Eagle s medium (DMEM), minimum essential medium (MEM), and Dulbecco s modified F12 medium (DMEM-F12), respectively, supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin at 5% CO 2 and 37ºC. Cellular Uptake Studies and Nanoparticle Calculations Sterile filtered ASNPs were added directly to the cell culture media of adherent cells in concentrations ranging from 1 to 60 nm. 48 hours after nanoparticle addition, the cells were washed 3 times in PBS buffer, collected, and counted using a Guava EasyCyte flow cytometer (Guava Technologies). To prepare samples for inductively coupled plasma mass spectrometry (ICP-MS) (Thermo-Fisher), the cells were dissolved with neat nitric acid at 60 C overnight. The Au content of the cell digest was determined by ICP-MS. Each cell sample was prepared in a matrix consisting of 3% HNO 3, 5 ppb Indium (internal standard), and Nanopure water. In order to extract the number of nanoparticles taken up by each cell, the number of nanoparticles must be calculated based on the concentration of Au found in the sample. This was done using

3 the molecular weight of Au and the diameter of the nanoparticle to calculate Au atoms per particle (6.78 x 10 4 atoms/particle). Once the number of particles was calculated, this particle number was divided by the cell count to determine the number of ASNPs per cell. Cell samples treated briefly (10 minutes) with ASNPs were prepared as above and used as controls for background subtraction in the case of ICP measurements to correct for any sticking of the nanoparticles to the cells and culture vessels during harvesting and preparation, or background ICP counts. All ICP experiments were preformed in triplicate and values obtained were averaged. To test the validity of calculated nanoparticle values, we analyzed Au NP solutions with known concentrations (Ted Pella, Inc., 7x10 11 particles/ml, 1.16 nm). Gold samples were centrifuged (13,000 rpm, 30 min) and then re-suspended in 25 μl of KCN (0.1M) for 3 hours to completely dissolve the particles. Concentrations of 290 pm, 120 pm, 29 pm, 12 pm, 4.6 pm, 1.8 pm, 500 fm, were analyzed by ICP-MS. Calculated values were similar to manufacturer concentrations (22.1% standard error). Surface Potential and Size Measurements ASNPs were diluted to 1 nm concentration in PBS buffer. Measurements were performed using a Zetasizer Nano ZS and disposable folded capillary cells (Malvern). Protein Adsorption ASNPs (final concentration 6 nm) were incubated both in serum-containing media and serumfree media for 24 hrs at 37º C. After this treatment, ASNPs were isolated from solution via three consecutive centrifugation steps (13,000 rpm, 20 min) and washed with PBS buffer to remove unbound proteins, and finally the Au NPs were dissolved with KCN (2.5 mm final concentration). A Quant-iT fluorescence protein assay (Invitrogen) was then used to determine the relative number of proteins in the solution. For the Quant-iT assay, KCN digested samples

4 were treated with a proprietary fluorescent reagent as described by the manufacturer, which when bound to proteins results in a fluorescent signal that is proportional to the amount of proteins in solution. After addition of the reagent, fluorescence was measured at room temperature using a fluorescence microplate reader (Fluo Dia T70, Photal) with excitation at 486 nm and recording emission at 570 nm. Estimation of the number of proteins in solution was calculated by comparing the fluorescence values of these unknowns to those obtained from a generated standard curve of bovine serum albumin (BSA) according to the manufacturer s recommendations and assuming an average protein size of 60 kd. Finally, the number of proteins per ASNP was calculated by dividing the number of proteins in solution by the initial nanoparticle concentration (6nM) prior to KCN digestion.

5 Supplementary Figure 1. Uptake as a function of the number of oligonucleotides per ASNP in C166 and HeLa cells (10 nm initial concentrations). 1. Frens, G. Nature-Physical Science 1973, 241, (105), Mucic, R. C.; Storhoff, J. J.; Mirkin, C. A.; Letsinger, R. L. Journal of the American Chemical Society 1998, 120, (48), Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A.; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Analytical Chemistry 2000, 72, (22),