**1. Introduction**

Since the very first example reported on nanometric mesoporous silica as a drug delivery material [1], many examples were subsequently reported including a broad number of chemical species. One of them includes biomacromolecules, which play a capital role in living beings, as they are responsible for biorecognition [2], signal transduction [3], and replication routes; hence, they are responsible for the adequate development of tissues and organs. Such is the importance of these species that even the immune system and hormone signaling are based on specific affinity interactions between biomacromolecules (BMs). From a therapeutic point of view, hijacking such ligands and receptors may be useful to regulate unbalanced systems, as well as develop new-generation therapeutic nanodevices in oncology [4,5] cancer immunotherapy [6–8], and gene therapy [9–11], among many others.

Unfortunately, most bioactive macromolecules are highly labile in vivo, as a self-regulation mechanism to avoid massive damages. Therefore, to use them, it is necessary to implement chemical modifications or to design vehicles capable of ensuring an adequate preservation and, hence, a long-lasting effect. Among known carriers, viruses have the best performance, although at the expense of having a grea<sup>t</sup> associated risk in handling and containment. Additionally, they are only suitable for nucleic acids (NAs), being useless for protein and peptide delivery. To overcome these limitations, nanoparticles emerged as promising vectors for nucleotides, as well as peptides and proteins. This is a consequence of two complementary aspects. On one hand, their size permits establishing intimate interactions with cell's membranes and the receptors therein. On the other hand, they exhibit the possibility to establish non-conventional interactions between particles, cargoes, and target cells.

Amongst all known materials, mesoporous silica nanoparticles (MSNs) arose as promising drug delivery platforms because of their outstanding biocompatibility, their degradability, and their grea<sup>t</sup> chemical and biological robustness. Moreover, the unique porous structure of MSNs permits establishing host–guest interactions of high interest for drug delivery purposes, as they allow creating protective environments for labile molecules. In addition to those, the current silica-based nanotechnology also permits creating particles with variable diameters [12,13], pore sizes [14], and structures [15], which permit fine-tuning the final application of the nanosystems, especially those intended to deliver big cargoes such as those reviewed herein. Moreover, MSNs and related hybrid particles with SiO2 coatings also permit easily tuning the resulting outer layers of nanosystems to enhance biochemical stability and, hence, to reduce side e ffects and potential toxicities [16–18]. Particle diameter is one of the most critical parameters for achieving a successful therapy. Typically, the accepted window of diameters for cancer treatment comprises particles in a range between 50 and 300 nm, in which the enhanced permeation and retention e ffect operates. However, depending on the final purpose of the nanosystem, such values may be narrowed. For instance, in cases where a superior tra fficking is desired, smaller particles would behave better, while, in nanosystems intended as biomolecule reservoirs, the diameter must unavoidably be increased. Regarding nanoparticle-based drug delivery, we recently reviewed how mesoporous silica-based nanosystems are suitable platforms to combine two or more chemical species, outranging the pharmacological profile of free species [19,20]. However, despite the possible therapeutic improvement, their e fficiency and long-term stability could be compromised if the BMs are not properly protected or fully exposed to white blood cells and immune systems. The scarce protection provided by most solid nanoparticles highlights once again the importance of MSNs [21] as platforms for the development of non-viral vectors and protein carriers, whose particular porous morphology can provide a protective environment for those labile molecules, although the typical porosity (2–3 nm) of MSNs may be tuned in order to host the biggest molecules [22] (Figure 1).

**Figure 1.** Main groups of therapeutic biomolecules that are possible to deliver using mesoporous nanosilica technology.

As introduced, the use of MSNs in biomedicine has a huge potential impact; in addition to acting as carriers, they also permit creating fancy structures with most functional nanomaterials. However, despite this versatility, the permeation of these materials into clinical trials is still limited [23,24]; this is not caused by poor biocompatibility, but rather by the impossibility of establishing reliable comparisons between di fferent systems, as accounted by Florence [25]. In fact, as can be observed from the growing number of in vivo experiments using MSNs carried out by many groups worldwide, it is logical to assume that they have adequate performance in living systems; thus, MSNs will hopefully soon reach clinical practice.

## **2. Strategies to Deliver Biomacromolecules with Silica Nanoparticles**

Loading e ffectiveness, cellular internalization, targeting, and cargo delivery are critical issues when developing a nanosystem with maximal therapeutic potential. As we previously mentioned, MSNs are ideal nanocarriers related to the load and delivery of biomolecules due to their unique properties, including shape, size, and surface chemistry [26]. Pore, channel, and cavity sizes can be modified in MSNs to increase the loading of therapeutic molecules [27]. The MSN surface can be functionalized with polyethyleneimine and poly-l-lysine or modified with targeting peptides or antibodies to improve cargo loading, cellular uptake, and endosomal scape rates [26]. The chemical and physical properties of MSNs are critical when designing a nanocarrier with therapeutic e fficacy in biomolecule loading and delivery. In the last few years, surface nanoscale topography gained special attention due to the possibility of controlling the interactions between molecules and cells, in the process of molecular loading and cell internalization [28,29].

The chemical functionalization of silica is easily achieved through condensation processes employing functionalized alkoxysilanes. For the functionalization of surfaces, this silanization must be carried out before template removal, while the modification of mesopores could be achieved either via condensation during the template-driven synthesis of MSNs or after surface modification and template removal. In this latter case, it is important to account that pore constriction may occur if the process is not properly controlled. For the preparation of hollow MSNs (HMSNs), the most employed method is the solid template etching of an internal core, typically from solid silica, onto which a mesoporous layer is created. Pore and surface modification can be achieved in a parallel manner as done with conventional MSNs, while, for the functionalization of the internal space, this must be done prior to the formation of the mesoporous layer. Onto this slightly modified MSN, it would be possible to create additional modifications by linking chemical species through conventional chemical reactions. With this strategy, it was possible to create a huge number of functional nanosystems with pore nanogates [30], sensitive bonds [31], recognition elements [5,19], and charged surfaces able to undergo electrostatic interactions.

One common strategy to combine BMs and NPs is surface functionalization; it can be achieved either via a chemical bond or via an electrostatic interaction between charged counterparts. On MSNs, surface grafting is usually accomplished via direct condensation of the remaining Si–OH groups with functional silane reagents; these are typically modified trialkoxysilanes with aliphatic chains bearing an additional functional group. For the bonding of peptides and proteins, the most common functional groups are amino and carboxylate for amide coupling and maleimides for thiol-mediated binding [32]. Additionally, to complement the direct coupling approach, there are also many di fferent bifunctional linkers available which are able to accomplish this task [33]. With regard to this coupling strategy, it is also important to remark the importance of controlling the bonding, which, if produced at the active region of proteins, may lead to inactivation. This is of particular importance when preparing antibody-targeted nanosystems [34,35] and sensors. On this topic, Landry and coworkers studied how the chemical linkage a ffected the specificity of cluster of di fferentiation 4 (CD4)-bonded proteins onto MSN against its target, the gp120 glycoprotein [36]. The authors proved that a conventional, unspecific, direct thiol–maleimide linkage behaved worse than a specific linkage placed far away from the active site.

The other approach for surface functionalization is electrostatic deposition. In this case, both nanocarriers and BMs need to establish strong electrostatic interactions through di fferently charged

functional groups. This is of particular importance for NAs, whose permanent negative charge permits strong interactions with positively charged surfaces and polymers such as polyethyleneimine or chitosan among many others. Insights into this strategy are available in References [20,37]. In the most advanced models, a rational deposition of alternating cationic and anionic layers—for instance, polyethyleneimine (PEI)–small interfering RNA (siRNA)–PEI–, also permitted developing multilayered nanosystems in which targeting elements could be added in the outermost layer. This design permits placing NAs in middle layers, obtaining additional protection against nucleases. Moreover, this strategy also enables pH-driven cleavage, which permits disassembling the system in endosomal environments, as a consequence of the proton sponge e ffect associated with polycationic substances [38]. The main drawback of the surface loading strategy is the absence of protection for BMs, which could be deactivated if exposed to blood (opsonization, enzymatic degradation, macrophage-mediated clearance, etc.) [39] or if not properly handled during processing (non-sterile material, accidental contamination, or physicochemical decomposition).

From a protective point of view, the use of pore-expanded MSNs [40] is the most convenient strategy, but only if the cargo is adequately retained until its final destination. To this end, pore sizes should be tuned to allow cargo hosting and, eventually, pore gates may be required [30]. The typical strategies to prepare enlarged-pore MSNs consist of either using large surfactants such as Pluronic or Brij [14] or employing swelling agents able to increase the diameter of the template cetrimonium micelles during the synthesis [41]. Additionally, the use of non-surfactant species, like tannic acid, was also reported as a pore-forming agen<sup>t</sup> [42]. Regarding NAs, it is also important to remark that raw pores of MSNs need to be chemically modified; otherwise, the negative charges of NAs and silanols would undergo a repulsive interaction that would hinder pore loading. This topic was previously visited by us and other groups in previous contributions [20,37]. In the case of proteins and peptides, this e ffect is not so relevant, as their isoelectric points are always closer to neutrality. In fact, most peptides behave as small molecules and can be loaded satisfactorily in most raw-pore MSNs.

In summary, the loading strategy must be carefully accounted for depending on the carried biomacromolecule. In this way, short peptides can be easily loaded within pores or grafted onto surfaces, while the delivery of bigger and highly charged molecules may su ffer from pore rejection if the mesopores are not properly conditioned [43]. Regarding NAs, their outstanding chemical stability permits creating either fancy layered structures or pore-loaded systems [44–46]. In addition to typical porous particles, the use of hollow mesoporous silica nanoparticles (HMSNs) also receives interest because of their additional enormous internal space. However, to use them as carriers, their mesopores and surfaces must comply with all requirements outlined for in-pore loading, i.e., su fficient diameter, favorable electrostatic environment, and adequate order to permit e ffective di ffusion processes.

In addition to cargo-related modifications, these kinds of nanodevices must also have additional modifications to increase colloidal stability and immune stealth to ensure an adequate tra fficking profile (Figure 2). In the case of pore-loaded nanosystems, this can be easily achieved with a common polyethylene glycol (PEG)ylation strategy. On coated nanosystems, it may not be necessary if the particles' coatings are naturally occurring biomolecules, such as those reviewed herein. In this case, although the integrity of supported molecules is not fully ensured, surface deposition is proven to be a synthetic advantage, as it greatly reduces the number of components and synthetic steps.

**Figure 2.** Blood compatibility, colloidal stability, and cell recognition are necessary on therapeutic nanosystems; otherwise, they would not reach their final destination.

## **3. Delivery of Proteins with Therapeutic E**ff**ect**

As introduced above, the effect and potency of therapeutic proteins rely on their physiological effect and behavior. For example, cytochrome c (Cyt c) triggers a caspase-mediated apoptosis, while immunoglobulins activate the immune system and induce cell destruction. Additionally, certain enzymes and growth factors may be useful to treat certain genetic diseases based on protein malfunction. Therefore, as there is no common therapeutic effect [47], the different approaches are discussed including pro-apoptotic, immunostimulating, enzymes, growth factors, and antibacterial proteins, according to the classification shown in Table 1 and Figure 3. For the interested reader, previously published outstanding revisions dealt with insights into protein loading and delivery with MSNs [48–51].


**Table 1.** Examples of therapeutic proteins delivered by silica-based nanocarriers.

