*3.1. Anticancer Proteins*

Historically, Lin's group was the first to describe the potential of MSNs to carry and deliver proteins. To achieve this, they focused on Cyt c, a small protein with a pro-apoptotic effect. In their contribution, they pioneered pore-expanded MSNs to allow protein hosting, highlighting the strategy to follow for the intracellular delivery of membrane-impermeable proteins [54]. Herein, they demonstrated that unmodified MSNs with 5.4-nm mesopores were able to host the globular 3.3-nm-width protein to produce effective intracellular delivery, although with no control. To improve this, Griebenow and coworkers evolved a system including a chemical bond able to retain Cyt c inside the mesopores. Their stimulus-responsive system was based on a redox-sensitive disulfide bond that linked Cyt c to thiol-modified mesopores [55], enabling an intracellular, glutathione-mediated release. Shang et al. also employed Cyt c as a model protein for MSN-based delivery, although, in their system, surface adsorption was preferred [56]. In this contribution, the authors did not evaluate the carrier efficiency or the stability of the protein but studied the loading capacity—protein activity—in relation to nanoparticle size. Their results showed that flatter surfaces—larger diameters—permit adsorbing more proteins and, hence, provide higher activities. Another example employing Cyt c was reported by Davis and coworkers, who studied the best-performing linker to connect the protein onto the particles' surface [57]. In their research, they systematically tested several custom-made linkers against the most critical parameters on optimal delivery for surface-grafted proteins: suitable charging capacity (>40 mV on ξ-potential), cationic character at acidic pH, ability to undergo endosomal escape, and capacity to permanently retain Cyt c immobilized on the surface. They found that MSNs modified with 1 mol.% primary amine were the only material able to satisfactorily accomplish all these tasks, providing a fantastic know-how for subsequent investigations into surface grafting. More recently, Choi et al. explored another possibility to deliver Cyt c, employing eroded MSNs with rougher surfaces and enlarged pores [58]. Herein, these matured MSNs permitted loading the Cyt c instead of obtaining the surface deposition that occurred onto particles bearing conventional (2–3 nm) pores. As a result of the

increased in-pore loading, the particles showed an enhanced release compared with both the free Cyt c and the nanosystem with higher surface deposition.

Apart from Cyt c, there were reported numerous anticancer proteins [52,53], albeit few examples in combination with MSNs. A relevant example on the topic was reported with concanavalin A, a lectin with anticancer and antibacterial properties [59,86]. Regarding the anticancer example, the lectin also proved to be an efficient targeting element against human osteosarcoma (HOS) and murine prosteoblast (MC3T3-E1) cells. In our system, MSNs were firstly loaded with the typical chemotherapeutic doxorubicin (DOX) and then coated with a polymeric layer linked to the silica surface through pH-sensitive linkers. To conclude, the concanavalin A (ConA) was grafted onto the surface through amide bonds. The resulting system was able to efficiently deliver the drug intracellularly, but only when the pH dropped enough to cleave the bis-acetal linker. The evaluation of the system demonstrated an enhanced killing effect when both species (ConA and DOX) were co-delivered. This suggested a potent adjuvant effect of the protein, which was able to drop the cell viability from 50% with DOX alone to almost 100%.
