*4.2. Liposome*

Liposomes are another type of exogenous TCL delivery platform. Due to the characteristic structure and composition of liposomes, the entrapment of hydrophilic cargo into the inner core of the liposomes, and additional lipid-mediated surface modification with functional moieties, are possible [131]. Based on these liposomal design strategies, Callmann et al. developed TCL-loaded liposomal spherical nucleic acids (Lys-SNAs) (Figure 4F) [120]. For their fabrication, TCLs from triple-negative breast cancer cells were encapsulated in the core of liposomes, while cholesteryl-modified immunostimulatory oligonucleotide adjuvants (CpG-1826) were immobilized on the surface. As described in the previous section, the oxidation of tumor cells prior to lysate generation using HOCl (OxLys) increases immunogenic aldehyde-modified antigens. After peritumoral administration into an EMT6 mouse mammary carcinoma model, OxLys-SNAs significantly increased the population of cytotoxic CD8<sup>+</sup> T cells, and simultaneously decreased that of myeloid derived-suppressor cells within the tumor microenvironment compared to Lys-SNAs and simple mixtures of OxLys. The enhanced therapeutic efficacy of the OxLys-SNA formulation was also indicated by antitumor activity, prolonged survival, and the inhibition of tumor regeneration. Therefore, the proper packaging and presentation of adjuvant and human-specific TCL-derived antigens into the liposomal structure is also an important design parameter for exogenous TCL delivery.

In addition to tumor-specific antigen delivery, leading to the maturation and activation of DCs, additional functions of liposomal carriers could facilitate immune modulatory responses. Won et al. [121] developed CO2-generating thermosensitive liposomes (BG-TSLs) that encapsulate melanoma-derived whole TCLs (Figure 4G). The lipid layers (a combination of DPPC/MSPC/DSPE-mPEG 2000) of these liposomal TCL carriers were fabricated using a thin lipid film hydration method [132]. Triggering TCL payload release by external near-infrared (NIR) irradiation increased anticancer responses through effective antigen presentation and maturation of DCs, T cell activation, and the proliferation of cytotoxic CD8<sup>+</sup> T cell populations. Moreover, CO<sup>2</sup> bubbles generated by the decomposition of the NH4HCO<sup>3</sup> co-payload enhanced the expression of pro-inflammatory cytokines, and suppressed tumor growth in tumor-bearing C57/BL6 mice models. Therefore, the combination of multiple cargo molecules with TCLs and the stimuli-responsive modulation of the liposomal architecture could be employed not only for in vivo DC activation, but also for therapeutic anticancer treatment with CpG-1826, which showed complete tumor remission after 100 days in 45% of the animals tested.
