Anisotropic Gold Nanoconjugates Bearing Immunoinformatically Designed T-Cell Epitope-Containing Peptides: Comparative Evaluation in THP-1 and Human Primary Macrophages.

Gold nanoparticles (AuNPs) are promising peptide carriers whose size and anisotropic morphology may influence their interactions with antigen-presenting cells. However, current knowledge of the morphology-dependent effects of AuNPs and the role of peptide design in human macrophage responses remains incomplete. Furthermore, most studies have been performed in THP-1-derived macrophages as the main in vitro macrophage-like model, despite human primary macrophages (HPMs) providing a more physiologically representative system. Anisotropic AuNPs with two morphologies, nanorods (AuNRs) and nanostars (AuNSs), each synthesized in two sizes, were characterized and conjugated to three influenza M2 T-cell-epitope constructs containing CD4+ (P1) and CD8+ T-cell epitopes (P2), individually, and both combined in a single multiepitope construct (P3), optimized for intracellular processing using a novel bioinformatics tool coupled to Predivac 3.0. THP-1-derived macrophages and HPMs were exposed to these nanoconjugates at a fixed particle number concentration of 10 pM for 24 h and assessed for mitochondrial activity, cell death, reactive oxygen species, endolysosomal acidification, proteasome activity, and cathepsin B activity. The identity of the nanoparticle carrier, defined by its geometry together with its specific surface chemistry, rather than the type of conjugated peptide, dominated the cellular response. AuNRs, particularly long rods, reduced mitochondrial activity, increased cell death, and enhanced bulk 20S proteasome activity in both models, even though at equal particle number they delivered 21- to 53-fold less gold mass and 6- to 12-fold less geometric surface area than AuNSs. The stimulatory effect induced by AuNRs-P1 and AuNRs-P2 on the proteasomal system may be associated with their specific peptide design features. By contrast, AuNSs preserved viability, maintained low oxidative stress, and produced strong acidification signals consistent with efficient trafficking to endolysosomal compartments; P2 and P3 conjugation further improved cytocompatibility and increased cathepsin B activity. HPMs were consistently more tolerant than THP-1 macrophages. The morphology-surface chemistry identity of the AuNP carrier is a key determinant of macrophage compatibility and of the relative engagement of the two degradative compartments assayed here. Functionalized AuNSs, especially P2- and P3-nanoconjugates, showed the best balance among biocompatibility, endolysosomal trafficking, and cathepsin B engagement. The proposed correspondence with MHC class I versus class II antigen-processing routes is a mechanistically informed hypothesis that remains to be further validated experimentally.