ABSTRACT: Background: Immune escape in the treatment by functionalized gold nanorods (Au NRs) should not be ignored. Whether CpG ODN, an immune adjuvant, can synergize with Au NRs to activate the immune response and its potential mechanism is not clear. Methods: The expression of CD8+ T cells, macrophage, NK cells, Th17, and Treg was detected by flow cytometry. Expression levels of IL-1, IL-6, IL-17, and TNF-α were assessed by ELISA. Subsequently, transcriptome sequencing analysis was performed to identify DEGs. GO, KEGG and PPI analyses explored the mechanism. The immune activation properties of antibody functionalization-associated Au NR were then compared. Results: Flow cytometry and ELISA analyses indicate that both Au NRs and CpG ODN promote the proliferation of inflammatory factors and immune activation. However, the CpG ODN-coupled Au NRs (Au NRs-C) demonstrated superior immune activation potential. Furthermore, Au NRs stimulate the expression of Treg, while Au NRs-C significantly inhibited this effect. This suggests that the conjugation of CpG ODN with Au NRs not only greatly enhances the immune activation but also compensates for their deficiencies in eliciting immune responses. Transcriptome sequencing uncovered DEGs mainly localized to immune and pro-inflammatory cytokine pathways. PPI analysis identified six hub genes: FOXM1, HMOX1, UBE2C, E2F1, PECAM1, and FCGR3A. Moreover, CpG conjugation with antibody-associated Au NRs enhances immune stimulation. Conclusion: Au NRs-C may upregulate the E2F1 and downregulate the PECAM1, which promotes MHC antigen presentation and Toll-like receptor-mediated immune processes while inhibiting CXCR receptor binding and immune negative regulation, thereby resulting in a stronger immune activation.
