*4.3. 3D Polymeric Gel*

The hydrogel-mediated co-delivery of multiple immune modulators has also been investigated. As an injectable vaccination platform, Song et al. developed poly(L-valine) (PEV)-based 3D peptide hydrogels for the co-delivery of melanoma-derived TCLs and a TLR3 agonist (Figure 5A) [133]. The sustained release of both tumor antigens and immune potentiators promoted DC maturation. The injected peptide hydrogel was able to maintain the localization of encapsulated TCLs at the in vivo vaccination site, and the expression of CD86 and MHC II antigens on DCs, and the CD8<sup>+</sup> T cell response, was significantly elevated compared to the administration of free TCLs or gels without the agonist molecule. Further tumor suppression also suggests that the formulation of peptide hydrogels encapsulated with TCL-derived tumor antigens and a TLR agonist could be utilized as a cancer vaccine platform.

**Figure 5.** Schematic illustration of TCLs and adjuvant co-delivery using exogenous delivery platforms. (**A**) After injection of TLR3 agonist and TCL-loaded mPEG-poly(L-valine) hydrogels, naïve DCs aggregate around hydrogel. (**B**) Natural component, β-glucan particle (GP)-based TCL and CpG delivery. All figures were reproduced with permission from Refs. [28,133]. **Figure 5.** Schematic illustration of TCLs and adjuvant co-delivery using exogenous delivery platforms. (**A**) After injection of TLR3 agonist and TCL-loaded mPEG-poly(L-valine) hydrogels, naïve DCs aggregate around hydrogel. (**B**) Natural component, β-glucan particle (GP)-based TCL and CpG delivery. All figures were reproduced with permission from Refs. [28,133].

The hydrogel-mediated co-delivery of multiple immune modulators has also been investigated. As an injectable vaccination platform, Song et al. developed poly(L-valine) (PEV)-based 3D peptide hydrogels for the co-delivery of melanoma-derived TCLs and a TLR3 agonist (Figure 5A) [133]. The sustained release of both tumor antigens and immune potentiators promoted DC maturation. The injected peptide hydrogel was able to maintain the localization of encapsulated TCLs at the in vivo vaccination site, and the expression of CD86 and MHC II antigens on DCs, and the CD8+ T cell response, was significantly elevated compared to the administration of free TCLs or gels without the agonist molecule. Further tumor suppression also suggests that the formulation of peptide hydrogels encapsulated with TCL-derived tumor antigens and a TLR agonist could be

A similar peptide hydrogel formulation has also been applied for the delivery and in vivo localization of multiple immune stimulants. mPEG-poly (L-alanine) (PEA)-based injectable peptide hydrogel could effectively encapsulate (1) melanoma-derived TCLs, (2) GM-CSF, and (3) dual immune checkpoint inhibitors (anti-CTLA-4/PD-1 antibody) during the spontaneous self-assembly of the polypeptide and subsequent gel formation via hydrophobic interactions [25]. Hence, persistent and synergistic DC activation by released TCL antigens and GM-CSF was achieved in C57BL/6J mice models with enhanced T cell responses. Especially, the augmented expansion of effector CD8+ T cells within the spleens and tumors of immunized mice by immune checkpoint blockade was observed. This hydrogel-based combination therapy showed superior immune modulation and anticancer efficacy compared to any single cargo delivery, demonstrating prolonged in vivo antigen-specific T cell immune responses. A similar peptide hydrogel formulation has also been applied for the delivery and in vivo localization of multiple immune stimulants. mPEG-poly (L-alanine) (PEA)-based injectable peptide hydrogel could effectively encapsulate (1) melanoma-derived TCLs, (2) GM-CSF, and (3) dual immune checkpoint inhibitors (anti-CTLA-4/PD-1 antibody) during the spontaneous self-assembly of the polypeptide and subsequent gel formation via hydrophobic interactions [25]. Hence, persistent and synergistic DC activation by released TCL antigens and GM-CSF was achieved in C57BL/6J mice models with enhanced T cell responses. Especially, the augmented expansion of effector CD8<sup>+</sup> T cells within the spleens and tumors of immunized mice by immune checkpoint blockade was observed. This hydrogel-based combination therapy showed superior immune modulation and anticancerefficacy compared to any single cargo delivery, demonstrating prolonged in vivo antigenspecific T cell immune responses.

Furthermore, cryogels (i.e., supermacroporous polymeric network obtained from the ice crystal formations through the steps of phase separation, crosslinking, and polymerization [134]) were also developed as a similar co-delivery platform for the in vivo administration of GM-CSF (DC enhancement factor) and CpG ODN (DC-activating factor) [135]. This cryogel-mediated vaccination platform effectively enhanced DC activation and leukocyte recruitment, and showed higher survival rates in melanomachallenged C57BL/6 mice models than bolus treatment with immunoactive factors. Furthermore, cryogels (i.e., supermacroporous polymeric network obtained from the ice crystal formations through the steps of phase separation, crosslinking, and polymerization [134]) were also developed as a similar co-delivery platform for the in vivo administration of GM-CSF (DC enhancement factor) and CpG ODN (DC-activating factor) [135]. This cryogel-mediated vaccination platform effectively enhanced DC activation and leukocyte recruitment, and showed higher survival rates in melanoma-challenged C57BL/6 mice models than bolus treatment with immunoactive factors.
