4.1.5. Adjuvant Activities of NPs

Some materials have shown potent adjuvant efficacy to stimulate cellular immunity and modulate immune responses. For instance, aluminum phosphate (AP) was discovered in 1926 as an adjuvant, and was later approved by the United States FDA [126,127]. Therefore, aluminum-containing adjuvants could also be used as cancer vaccines by antigen adsorption via electrostatic attraction and ligand exchange. In particular, CpG-loaded AP NPs coated with B16F10 tumor cell membranes have been developed for cancer vaccination in melanoma models [35]. Again, the surface-incorporated cancer cell membranes enhanced the colloidal dispersion of AP NPs and functioned as native tumor antigens. The dual functions of the AP-mediated adjuvant effects and immunogenicity of antigens effectively mDCs activation, improved lymph node targeting, and facilitated strong tumor-specific cellular immune responses after subcutaneous injection in mice.

Chitosan, a cationic polysaccharide, is also widely used as a vaccine delivery vehicle due to its adjuvant efficacy to promote IFN secretion in mature bone marrow-derived cells (BMDCs), and thus, enhances antigen-specific Th1 responses [128]. Chitosan adjuvants delivered to DCs could induce mitochondrial stress and generate ROS. Subsequent activation of the cGAS-STING pathway triggers the production of type I interferons, and further DC maturation occurs. In addition to the adjuvant effect of chitosan, the Man-based surface functionalization of core chitosan NPs (Man-CTS NPs) facilitates the targeting efficacy of TCL delivery to APCs by binding to Man receptors located on DC membranes [22]. This Man coating also enhances the in vitro bone marrow DC uptake of antigens in TCLs through receptor targeting [129]. Therefore, treatment using B16 melanoma TCL-loaded Man-CTS NPs augmented DC maturation and the related antigen presentation, indicated by the enhanced expression levels of surface markers (i.e., MHC I, MHC II, CCR7, CD80, CD86, and CD40) in vitro and in vivo. An elicited adjuvant effect and T cell priming were further observed with the increased proliferation of both CD8<sup>+</sup> and CD4<sup>+</sup> T cells, and the upregulated expression levels of serum IFN-γ and IL-4, confirming in vivo T cell activation in melanoma mice models. Vaccination efficacy and therapeutic effects of TCL-loaded Man-CTS NPs were proven by tumor growth inhibition and reductions in tumor weight.

Additionally, the neurotransmitter dopamine (DA) has also been used for the immune system activation of effector T cells and the suppression of regulatory T (Treg) cells by reacting with DA receptors. DA activates NF-κB to upregulate pro-inflammatory cytokines and chemokines (e.g., IL-6, IL-1β, IL-18, CCL2, and CXCL8) [130]. Wang et al. synthesized polydopamine (PDA)-based NPs covalently conjugated with colorectal cancer TCLs (TCL@PDA NPs) by the interaction between catechols in DA and the amine/thiol groups of antigens in TCLs [24]. PDA-based NPs showed potential as an antigen carrier, exhibiting (1) PDA-mediated pro-inflammation, with increased secretion of IFN-γ and TNF-α, and (2) DC maturation, with the enhanced expression of MHC II and secretion of Th1-related

cytokines. In a C57BL/6 mouse model, three (day 4, 10, and 18 after cancer inoculation) subcutaneous vaccinations with TCL@PDA NPs significantly increased the subpopulations of CD4<sup>+</sup> and CD8<sup>+</sup> T cells in the spleen and LNs, as well as the memory T cell response. Therefore, both in vivo antitumor efficacy and tumor prevention effects were sufficiently achieved by the combination of PDA and TCLs.
