*3.2. Immunostimulating Proteins*

Immunostimulation is one of the most popular strategies for improving cancer treatment, as it promises to recruit the patient's immune system to fight tumors. However, any indiscriminate activation may lead to disastrous results. Therefore, there is interest in developing immunostimulant nanosystems able to induce local immune responses thanks to the enhanced permeability and retention (EPR) effect. MSN-based immunotherapy also has an additional advantage, as these particles were proven to be efficient coadjutants [87–89]. Despite these promising features, the delivery of immune-regulating proteins with silica-based carriers is still in its infancy.

Regarding already reported examples, proteins and MSNs can be combined via either surface deposition—grafted or not—or loading into enlarged pores, although those are not the only options. One such exception was the contribution by Lim et al. who delivered immunoglobulin (IgG) to HeLa cells employing a very special silica particle [60]. These were called unconventional perforated HMSNs. Those particles were able to efficiently deliver large membrane-impermeable cargoes, although without any study on immune response. However, their contribution established the basis for local immunostimulation, opening the way to new therapeutic strategies. In more recent examples, several research groups studied immune inductions in mice using nanoparticles. Along this line, Wang, Ito, Tsuji, and coworkers reported a series of articles which firstly studied the immunostimulating behavior of HMSNs [87–89] in murine models, and then studied how the surface modification of such HMSNs with the T cell-dependent antigen, chicken ovalbumin (OVA), stimulated the overall response [61]. The authors paid special attention to which markers were upregulated when treating the animals with these devices [62]. They found a four-action pathway: an anticancer effect through the use of HMSNs themselves, an effector memory on the CD4+ and CD8+ T-cell population, an overexpression on T helper 1 (Th1) and Th2 cytokines, and an enhanced secretion of immunoglobulin antibodies [62]. Employing another silica nanostructure, a multi-shelled dendritic mesoporous organosilica hollow sphere (DMOHS), Yang et al. also obtained similar stimulation patterns when they employed OVA-loaded particles [63]. Herein, a parallel upregulation on CD4<sup>+</sup>, CD8<sup>+</sup>, and Th1 immunoproteins was also reported, characterized by the secretion of interleukin-12 (IL-12), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). More recently, Cha et al. evolved this system by adding an additional immunostimulating entity. In their model, large-pore MSNs (20–30 nm) were sequentially loaded with OVA plus an additional "danger signal", an agonist for Toll-like receptor 9 (TLR9) [64]. As a result, an increased immunostimulation was obtained, which again suggests the importance of combined therapies for the development of more effective cancer treatments and vaccines. More recently, these authors implemented their immunostimulant nanosystem by designing a combination of these MSNs embedded in large chemokine-loaded mesoporous nanorods suitable for injection [65]. With this system, the authors overcame the limitations of the intravenous

dosage of their previous nanosystem, as, with this approach, they could e ffectively recruit and mature the dendritic cells in order to achieve a more e fficient cancer vaccination. Their final formulation demonstrated a significant tumor progression reduction together with grea<sup>t</sup> survival rates.

In addition to the typical strategies to link proteins onto nanoparticles, Niu et al. reported another possible approach for protein delivery. They designed di fferent porous systems with variable cavities by controlling a core–shell assembly between solid silica nanoparticles. In their designs, core particles had a preset size, while encircling particles had smaller but variable diameters. The resulting disposition created di fferent interparticle voids able to host di fferent sized proteins [66]. The resulting nanostructures showed variable roughness (14, 21, and 38 nm, respectively) that permitted satisfactorily loading a wide variety of proteins, such as Cyt c, monoclonal rabbit antibodies (IgG), and even antibody fragments (horseradish peroxidase (HRP)-linked anti-rabbit IgG antibody), preserving in all cases their activities, as demonstrated by surface plasmon resonance. From a therapeutic point of view, the most interesting model was the 38-nm hydrophobically modified rough (non-porous and core-shell) silica nanoparticles (RSNs), which were able to deliver the anti-pAkt antibody into MCF-7 breast cancer cells. In this case, the therapeutic e ffect occurred because the antibody created a significant reduction of cell proliferation, together with a downregulated expression of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein.

In addition to immunogenic proteins, another interesting therapeutic possibility enabled by nanotechnology is non-viral-based vaccination [90,91]. Therefore, this could be achieved if antigens are delivered to immune cells, thereby avoiding a general immune response. One of the first examples of vaccination employing silica nanoparticles was reported by Guo et al., who employed HMSNs to deliver the open reading frame of the porcine circovirus type 2 (ORF2) protein using a murine model [68]. In this case, the nanovaccine was prepared via direct adsorption of the protein onto raw nanoparticles. In mice, the MSN-based delivery of ORF2 induced overexpression of the typical markers: IFN-γ, Th1, CD4, and CD8, suggesting an immune activation. This strategy was also successfully employed to create other specific MSN-based non-viral vaccines for mice: (1) against *Schistosoma mansoni* employing homogenates from the parasite [69]; (2) against porcine enzootic pneumonia by using a recombinant heat-shock protein 70 (HSP70) antigen fragment (HSP70212–600) from *Mycoplasma hyopneumoniae* [70]; (3) against enterohaemorrhagic *Escherichia coli* by using a recombinant fragment of filamentous immunogenic protein from enterohaemorrhagic *E. coli* (EspA) protein [71]; (4) against the pathogenic fungus *Paracoccidioides brasiliensis* employing the antigenic protein rPb27 [72]. In light of these results, it is noteworthy to highlight that these nanoparticle-based vaccinations permit immunizing against di fferent types of pathogens, such as parasites, fungi, or bacteria, with a better profile than the free antigen or other known pharmacological formulations in which the delivery toward macrophages is not so e fficient.
