4.1.4. Inorganic Templates for TCL Delivery

In addition to polymeric NPs, inorganic porous particles, such as calcium carbonate (CaCO3) and mesoporous silica NPs (MSNs), have also been used as templates for the encapsulation of proteins and peptide antigens. Lybaert et al. [23] utilized CaCO<sup>3</sup> particles covered with a polymeric TLR7/8 agonist (CL264) to encapsulate TCLs. CaCO<sup>3</sup> particles with highly porous inner architecture showed a high loading capacity for macromolecules via surface adsorption and encapsulation into the inner core. Surface coating with polycations of the copolymer of N -(hydroxypropyl) methacrylamide (HPMA) and N-(3-aminopropyl) methacrylamide (APMA) modulated the surface charges to adsorb the TLR 7/8 agonist by the combination of electrostatic interaction and physisorption. Additionally, TCLs were prepared from the Lewis lung cancer cell line expressing OVA antigens, coprecipitated with CaCl<sup>2</sup> and Na2CO<sup>3</sup> during the fabrication of CaCO<sup>3</sup> particles, and were encapsulated into the core.

The delivered OVA-containing TCLs using TCL-TLR-CaCO<sup>3</sup> particles resulted in the cross-presentation of OVA by DCs after the migration of the particles into phagosomes and fusion with acidic lysosomes [123]. The results of the co-delivery of tumor-associated antigens using TCLs and the TLR7/8 agonist indicate the higher efficiency of cross-presentation and in vivo antitumor responses via enhanced immunogenicity, compared to any single treatment.

Since TLR is one of the PRRs in DCs [124], this co-delivery strategy using TCLs-TLR-CaCO<sup>3</sup> particles could (1) activate PRRs by pathogen-associated molecular patterns (PAMPs) and DAMPs derived from necrotic cells (i.e., TCLs), and (2) upregulate antigen presentation by the additional efficacy of the TLR agonist as a potent activator.

A similar surface coating was also used to fabricate cancer cell membrane-coated MSNs [119]. Likewise, the chemotherapeutic drug doxorubicin (DOX) was entrapped in the inner porous structure of the MSNs (i.e., DOX-MSNs), and membrane fragments from LNCaP-AI prostate cancer cell lines (CMs) were then adsorbed onto the DOX-MSNs (i.e., DOX-MSN-CM) (Figure 4D). Along with the induced apoptosis of prostate cancer cells, the co-administration of DOX and CMs using MSNs significantly suppressed tumor growth in LNCaP-AI tumor models.

Recently, liquid metal (LM) has also been utilized as a template core for the development of a nanovaccine for tumor prevention [116]. In this study, CMs derived from 4T1 murine breast tumor cells were coated onto mPEG5000-SH-modified eutectic gallium– indium LM NPs (Figure 4E). As well as the antigenic efficacy of CMs and the immune adjuvant effect of LM, the additional photothermal conversion efficacy of LM NPs irradiated by an 808-nm laser facilitated local inflammation, and the subsequent recruitment of APCs, by the increased secretion of pro-inflammatory factors (i.e., IL-6 and TNF-α) and metal-induced NF-kB immune activation pathways [125]. In addition to the effective in vivo delivery of antigens to lymph nodes, three vaccinations within 15 days before the inoculation of 4T1 tumor cells in a mouse model also indicated the significant tumor prophylactic efficacy of CM-coated LM NPs with laser irradiation.
