4.1.1. Design Parameters for TCL Carriers

The most representative TCL delivery platform comprises nanoparticle (NP)-based carriers, which use several types of materials decorated with functional moieties to boost their delivery efficacy. The encapsulation of TCLs in the NP core can protect cargos from degradation during in vivo circulation, and regulate their release. These particlebased carriers can also be easily modified with functional ligands or molecules on their surface [98,99]. The efficiency of antigen-containing TCLs in draining lymph nodes can be influenced by the characteristics of the template particles, such as size, morphology, and charge.

For example, the efficiency of nano-sized polystyrene particles for activating APC subsets was higher than that of micro-sized particles in terms of cellular uptake [100]. NPs can easily infiltrate cells, and their resident particle populations in lymph nodes are three fold larger than those of micro-sized particles. Thus, a potential T cell immune response can be effectively induced by DC maturation. Moreover, this size-dependent immunogenicity was also observed in vaccination efficacy against tumors [101]. The delivery of human papillomavirus peptides using 40–50 nm Ag NPs resulted in higher uptake into DCs in draining lymph nodes, in vivo localization in C57BL/6 mice models, and immunological responses. Protection in tumor challenge models and the clearance of established tumors was also found.

In terms of the morphology and geometry of particulate carriers, the spherical shape of NPs exhibits (1) reduced adhesion to vessel walls, and longer circulation time [102], and (2) facile ligand conjugation onto larger surface areas. Spherical NPs with surfaceconjugated ligands can be fully enveloped by target cellular membranes via strong ligand– receptor interactions, and consequently facilitate receptor-mediated endocytosis [103]. Spherical NPs could overcome a minimal membrane binding energy barrier, resulting in low free energy change for internalization into target cells [104]. Shape-dependent immune adjuvant efficacy was also observed in the delivery of AuNPs coated with virus envelope proteins (VEPs) [105]. The in vivo inoculation of these NP-VEPs into mice resulted in shapedependent cytokine production in DCs. Rod-shaped NPs induced pro-inflammatory IL-1β and IL-18 production by activating the inflammasome-dependent process as adjuvants for eliciting immunity. In contrast, the same antigen delivery system using spherical or cube-shaped NPs induced the secretion of other types of inflammatory cytokines, including TNF-R, IL-6, IL-12, and GM-CSF.

An optimal surface charge and charge density of NPs is also required in order to increase the duration of blood circulation and prevent their loss to untargeted regions [106]. In general, positively charged NPs interact more efficiently with negatively charged cell

membranes, and higher cellular uptake occurs [107]. A previous study reported chargedependent NP uptake by 3T3 fibroblasts [108], indicating that the cellular internalization of trimethylammonium-coated AuNPs with positive surface charges was faster than that of negatively charged phosphonate-coated particles. The interaction with various in vivo protein components and delivered NPs resulted in the formation of a protein corona, which might reduce NP uptake regardless of the charge of the NPs. Moreover, a higher concentration of positively charged NPs (>5 nM) caused oxidative stress and cell death. Thus, the charge property of NP carriers should be also optimized to improve colloidal stability and interaction with target cells, and, therefore, effective exogenous delivery of antigen molecules.
