*3.3. Enzymes*

The deregulation of normal enzymatic levels has a fundamental role in many diseases. For example, toxic or metabolic syndromes are a direct consequence of this protein malfunction. In cancer, the deregulation of homeostasis is also a consequence of an abnormal expression of proteins and enzymes. However, the use of enzymes in therapy clearly goes beyond cancer treatment based on the delivery of cell-damaging proteins. Unfortunately, like all proteins, enzymes usually do not nicely tolerate systemic administration unless properly modified [92]. Such is the case of the p53 anticancer protein capsule designed by Zhao et al. [93] or the collagenase capsule reported by Villegas et al. [94], which performed best only when they had a degradable protective shell. With regard to the enzymes delivered by MSNs, it is important to remark that most examples are located at the surface; otherwise, the di ffusion of both substrates and products may be di fficult. One of the most studied examples is carbonic anhydrase (CA), which, despite not having a significant therapeutic e ffect, was widely employed as a model for in-pore loading approaches [73,74]. The advantage of CA as a model is a direct consequence of the facile determination of its remaining activity, as both substrate (CO2) and final product (HCO3 −) are small enough to di ffuse through the pores with freedom and can be easily determined.

Regarding therapeutic models, the mitigation of toxic syndromes caused by genetic disorders is one of the most promising research fields. One interesting example was reported by Xu et al. [75], who employed MSNs to deliver β-galactosidase (β-Gal) to treat Morquio B syndrome. This disease appears when the enzyme is not able to properly cleave the glycoside bond on oligosaccharides, thus producing the accumulation of substrates. In this case, contrary to other models in which the enzyme was located on the surface, the authors decided to load it into the mesopores to obtain additional protection and long-term stability. In this case, the significant size (119 kDa) of β-Gal made it mandatory to prepare ultra-large-pore MSNs. For this, the authors created core–cone structured MSNs with dahlia-like mesopores that were able to host, preserve, and deliver catalytically active β-Gal to N2a cells. In this case, the large size of mesopores, together with the small sizes of both enzymatic substrates and products, permitted supporting the enzyme within the pores; however, in other cases, in which the substrates have di ffusion barriers because of the size or nature, this approach may not be so convenient.

With regard to supported proteins, Mou and coworkers reported the use of two antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx), to intracellularly deplete reactive oxygen species (ROS). In this case, the di fferent nature of possible substrates demanded an adequate exposure of proteins on the nanosystem; otherwise, they would not be able to prevent oxidative stress. In their first example, the authors developed a mesoporous silica-based multifunctional nanocarrier for SOD [76]. This device employed an Ni<sup>+</sup>2-chelated nitrilotriacetic acid (Ni-NTA)-modified silane, which was able to connect through a histidine moiety to a TAT-containing peptide sequence bound to the enzyme. At this point, the authors denatured the enzyme with urea in order to have only intracellular refolding and to avoid extracellular ROS depletion. The resulting system was able to internalize into HeLa cells and, therein, refold the enzyme into its active form and reduce the oxidative stress induced by paraquat, a well-known superoxide anion generator. More recently, the authors implemented the antioxidant performance of this model by employing two di fferently loaded nanodevices: one with SOD and the other with GPx [77]. With this combination, the authors found a complementary synergic e ffect in which the co-delivery of both antioxidant proteins improved the e ffect of single treatments.

Another interesting example, reported by Han et al., focused on the delivery of human proteasomes to delay tau aggregation associated with Alzheimer's disease [78]. In this model, the authors employed the same building strategy reported by Mou's group: Ni-NTA able to bind histidine moieties from active human 26S proteasomes isolated from a HEK293-derived cell line. Again, the system proved to deliver proteasomes without a significant proteotoxic e ffect associated with its enzymatic activity. At this point, the authors claimed that their system was not intended to permeate the blood-brain barrier, although it could be implemented onto permeable systems, opening the way to novel strategies to prevent and treat Alzheimer's.
