The tandem repeats of peptides, incorporated onto this AuNV design, have shown improved vaccination efficacy in non-gold particle systems [18, 19]. Furthermore, the simple bottom-up conjugation design can allow effective delivery of large doses of vaccine peptides and thus improve

immunogenicity of the vaccine DAPT clinical trial antigen peptides. Here, we evaluated the high-peptide density AuNVs through three steps: synthesis and characterization, AuNV uptake by dendritic cells, and functional in vitro immunologic assays. Figure 1 Schematic of gold-based nanovaccine design synthesis. The AuNPs were coated with self-assembled monolayers of 5000-MW PEG-SH. The AuNPs were subsequently conjugated with the desired peptides using EDC and sulfo-NHS as linkers. Methods Reagents All of the polyethylene glycol (PEG) products were purchased from NanoCS (New York, NY, USA). The citrate-stabilized gold colloids were purchased from Ted Pella (Redding, CA, USA). All of the buffers and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), Thermo Scientific (Waltham, MA, USA), and Invitrogen (Carlsbad, CA, USA). The peptides were purchased from Genemed Synthesis (San Antonio, TX, USA). The JAWS II cells and media were purchased from ATCC (Manassas, VA,

USA). Two-step AuNV synthesis First, carboxyl-PEG-thiols were added to a 30-nm gold colloid solution (2 × 1011 particles/ml) with an end concentration of 5 μM and incubated for 24 h. The solution was raised

to 0.1 M NaCl, 10 mM sodium phosphate, and 0.1% click here Tween 20. The excessive PEG molecules were removed from the AuNP solution by three centrifugation-washing steps at 7,000×g for 20 min with phosphate-buffered saline (PBS). The final particle pellet was diluted with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. EDC (4.25 mg) and sulfo-NHS linker (6.4 mg) were added to the particle-MES solution and incubated for 15 min at room temperature. The excessive linkers were removed from the solution enough by centrifuging in a 10,000 molecular weight cutoff filter at 2,000×g for 15 min and diluting the particles with PBS. The peptides (50 μg) were then added to the particles per milliliter of solution, and the mixture was incubated for 30 min, 1 h, 2 h, and 24 h at room temperature. Varying the incubation time was for optimization of the conjugation scheme. Hydroxylamine (10 mM) was added to quench any unbound EDC/NHS for an additional hour. The peptide-coated particles were then centrifuged and washed three times with PBS. After the final PBS wash/centrifuge cycle, the supernatant was removed, and the particle pellet was re-suspended in 200 μl of PBS. The sample was sonicated and stored at 4°C until used.