Trp-2 AuNVs remained in solution when ethanol (0.2% v/v Tween 20) was added to the tubes due to the decrease in polarity of the solvent and the addition of surfactants (Additional file 1: Figure S8). Thus, AuNV particle behavior in solution is dependent on the peptide properties. Having high peptide density on AuNVs is important for vaccine function because the peptide-coated nanocarriers collect in the endosomes and can mimic the size of pathogens, stimulating DC maturation. The induction of DCs to mature and to present tumor antigens is crucial for engineering a successful vaccine. This stimulation by nanomaterials has been shown by Moon et al. to cause DCs to induce large amounts of cross-presentation for stronger and sustained anti-tumor immune responses . Cross-presentation is very important for CTL stimulation because it is required to allow peptides to enter the MHC class I (PRIMA-1MET order cytosolic) pathway from the MHC learn more class II (endosomal) pathway. By using MHC class I peptides, DC-to-splenocyte ELISPOTs can be used to evaluate the extent of cross-presentation. Additionally, the assay itself is of interest because it can screen VX-661 cell line large numbers of nanovaccines in vitro, simulating the process of antigen presentation and preventing extensive use of animals. Once the AuNVs enter the endosomes, it is critical that the peptides can come off the particles
and enter the MHC class I pathway. Therefore, the conjugation optimization of conjugation duration and schemes is a key for an effective AuNV. From the
optimization results, we concluded that the 1-h conjugation time was most effective. We hypothesize that the peptides link linearly during the 1-h conjugation but will begin to cross-link transversely via peptide side groups by 2 h. The non-linear cross-linking could disrupt the peptide sequence or presentability, thus lowering the efficacy and size of those AuNVs. As for the method optimization, the buffers used for the conjugation process cause a significant impact on the AuNV efficacy. MES buffer has a pKa of 6.15, which is within the range for EDC coupling to check details carboxyl groups generating O-acylisourea [28, 29]. Sulfo-NHS was then added to replace the O-acylisourea to form semi-stable amine-reactive NHS esters. Amine binding to the NHS esters reacts better at neutral to higher pH . Thus, switching to PBS at pH 7.4 prevents excessive self cross-linkage. Furthermore, the one-step (MES) method allows better carboxyl activation and a higher chance of extra linkages or cross-linkage, but it can also cause excessive cross-linkages from the side changes of the peptides, which can lower the efficacy of the vaccine peptides. Conversely, the two-step method (MES-PBS) allows less side chain linkage but lowers overall peptide linkage. From the results, the one-step method AuNVs were significantly better at stimulating CTLs than the two-step method.