The one-step method

used lower amounts of EDC/NHS, and the excess linkers were not washed away. In this scenario, the process remained in the MES buffer at pH 6. We hypothesized that the one-step method would be more effective at forming chained peptides, and experimental results support that claim. Using the DC-to-splenocyte IFN-γ assay, the PS-341 ic50 one-step MES buffer method (231 SFC) was significantly more effective in inducing IFN-γ secretion than the two-step design (182 SFC; p = 0.004) (Figure  5B). Therefore, the following experiments used the one-step conjugation scheme. Comparing efficacies of DNA linker and PEG linker AuNVs Since PEG is non-degradable, using PEG as linkers raises concern of whether the peptides were released from the AuNPs, which is critical for MHC class I and II loading. To address how peptides are released from the AuNVs following conjugation with the PEG linkers, we examined the degradation of FITC-labeled AuNVs by cell proteases from the JAWS II lysate, an immortalized BMDC cell line. The FITC-labeled AuNVs were incubated with the cell lysate for 24 h. Then, the mixture was centrifuged to remove all the particles and large cell debris.

PKA activator The peptides that came off the particle would then be in the supernatant. Both the OVA and gp100 AuNV showed a twofold increase in the FITC fluorescent levels in the supernatant post-lysate exposure, supporting the hypothesis that cell proteases contained in DCs could remove antigenic peptides from the surface of AuNPs (Additional file 1: Figure S5). By replacing the non-degradable PEG linkers with poly-T DNA spacers, selleck screening library DNases in the cells would be expected to degrade the spacer and to release the peptides from the particle surface. This release mechanism should improve the immunogenicity of the AuNVs. The DNA spacer (11 nt) had a thiol modification on the 5′ end and a primary amine on the 3′ end. The thiol forms dative bonds with AuNPs in a manner similar to that of PEG-SH. DNA spacers presented amines on the AuNP surface instead of carboxyl groups. Thus, the carboxyl activation step was removed

because there were no longer any carboxyl groups on the particles. This change, however, did decrease the peptide conjugation yield. As previously mentioned, the BMDCs were incubated with the AuNVs to minimize non-specific IFN-γ secretion, Then, the loaded BMDCs were exposed to antigen-specific splenocytes, i.e., OT-I for OVA peptides and pmel-1 for gp100 peptides. When the AuNV-loaded DCs were exposed to OT-I splenocytes, the AuNVs (134 SFC), maximum dose of 10 μg/ml, showed significant improvement in IFN-γ secretion compared to the free-peptide control (10 μg/ml; 103 SFC) (Figure  6A). Although the improvement of the OVA AuNVs compared to the OVA free peptides was not as large relative to the gp100 AuNV samples, both tested AuNVs showed a significant improvement in the splenocyte response compared to the free peptides (p < 0.01) (Figure  6B).