## 3 Nanostructured Systems for Cancer Immunotherapy
An alternative delivery vehicle for imiquimod has been developed by [PERSON] et al. [76], who created pectin-based hydrogel nanocapsules for treating melanoma. This study revealed that the fabricated nanosystem allowed drug penetration in all skin layers and led to a higher cytotoxic effect of imiquimod in tested tumor cell populations, demonstrating promising potential for improving immunotherapy for this disease. Imiquimod activity can also be improved by its encapsulation into nanoemulsions, as demonstrated by [PERSON] et al. [77]. The researchers have tested the nanoformulation against cervical cancer, proving its augmented antitumoral effect compared to free drug attributed to the combined autophagy and apoptosis mechanisms. Several nanoformulations were also proposed for the delivery of resiquimod. For example, [PERSON] et al. [78] proposed encapsulating this TLR agonist into \(\beta\)-cyclodextrinrin nanoparticles to ensure its efficient delivery to tumor-associated macrophages. The authors even combined this strategy with the administration of immune checkpoint inhibitor anti-PD-1, obtaining synergistic results in terms of immunotherapy response rates. Differently, [PERSON] and colleagues [79] have created a resiquimod delivery system based on PLGA/mPEG-PLA nanoparticles that are internalized in dendritic cells and macrophages, which further activate anticancer immune responses. Based on in vivo tests performed on mice, the nanoformulation was proven effective for targeting lymph nodes and improving cancer immunotherapy outcomes. A different approach for inducing strong immune responses involves the targeted delivery of specific nucleic acids to tumor sites. Such gene delivery systems ensure the internalization of the therapeutic cargo, further reflected in the activation of cellular and humoral immune responses and eventual tumor destruction (Figure 5) [39]. Embarking on this possibility, [PERSON] et al. [80] proposed the utilization of guanosine- and uridine-rich single-stranded RNA (GU-rich RNA) as the therapeutic agent, being recognized as an agonist of TLR7 and TLR8. In this respect, the researchers prepared a nanostructured hydrogel assembly of GU-rich RNA/DNA capable of sustained release. The delivery system was successfully internalized in murine dendritic cells and generated high immunostimulatory activity, and is thus considered a valuable adjuvant in cancer immunotherapy. Figure 4: (**A**) Synthesis and dual pH-responsive (or enzymatic) degradation of amphiphilic poly (esteracetal) block copolymers. (**B**) Schematic illustration of the concept using amphiphilic poly (esteracetal) block copolymers and their derived micelles for the pH- and lipase-responsive delivery of immunodrugs. Reprinted from an open-access source [15]. Compared to passive delivery vehicles, stimuli-triggered nanosystems allow for more accurate spatiotemporal control over TLR7/8 agonist activity in response to specific tumor microenvironment (TME) aspects, such as hypoxia, acidity, and hypoglycemia. Nanobiotechnological systems can selectively release their cargo due to temperature, pH, or redox potential changes and cross biological barriers to enhancing immunogenic cell death in therapies like chemotherapy, radiotherapy, and magnetic hyperthermia [10; 70; 81]. For example, [PERSON] et al. [82] based their therapeutic approach on the pH-responsive character of a novel-designed nanosystem. Specifically, the researchers created nanometric self-assembling micelles made of newly synthesized fatty acid-protic ionic liquids (FA-PILs) and vitamin E for the delivery of initiumod. The developed nanosystem allowed the controlled release of the TLR agonist in cancer tissues, which were activated by the acidic conditions of the TME and hold promise for skin cancer treatment. [PERSON] and colleagues [83] have proposed a different pH-responsive delivery platform. The authors suggested using polymeric nanoparticles to transport a novel imidazoquinoline-based TLR7/8.