*4.4. Growth Factors*

As in many other cases, peptides could also have anabolic properties. These features make them interesting candidates for their use as cargoes in new-generation therapeutic devices aimed at tissue growth induction or hormone therapy. The first example in this field, reported by Lin's group, employed MSNs to intracellularly deliver cyclic adenosine monophosphate (cAMP). To obtain chemical responsiveness, the authors modified the MSNs with a boronic acid –B(OH)2 moiety able to coordinate glucuronic acid moieties. The mesopores were then loaded with cAMP and closed through glucuronate-modified insulin nanocaps [125]. The resulting system was sensitive to glucose, as it could be disassembled by chemical displacement. As a result, this system permitted regulating hyperglycemic levels through a dual release mechanism: Insulin to trigger glucose assimilation by liver, and cAMP delivery to induce glycolysis. Unfortunately, this proof of concept was not further evaluated in vivo.

More recently, Sun et al. revisited insulin delivery with MSNs. To do so, they developed a glucose-sensitive nanodevice employing rod-like MSNs and a polymer shell able to change its conformation upon the presence of glucose [126]. In their investigations, the authors optimized the composition of this polymeric shell by using three di fferent monomers: *N*-isopropylaminomethacrylate (NIPAM) to provide the needed morphological shift, 3-acrylamidophenylboronic acid as a glucose-sensitive fragment, and dextran maleate as an optional hydrophilic component. The resulting system could be loaded with insulin at low temperatures (4 ◦C), in which the polymer adopted an expanded conformation and closed upon heating (37 ◦C). Both reported compositions showed e ffective insulin release when glucose was present; however, when pH was below 7, the polymer was not able to shift morphology, avoiding insulin release. Although only in vitro tests were reported, the possibilities of this model are considerable, as the incorporation of dextran may favor colloidal stability and enhance biocompatibility, as suggested by the high cell viability values obtained. Insulin delivery was also investigated by Zakeri-Siavashani et al., who employed SBA-15 mesoporous microparticles [127]; however, therein, the authors profited from the bigger pore size provided by these particles to achieve higher loading and more sustained release. Although their system showed a nice insulin release profile, the typical bigger sizes of SBA-15 particles (generally above 300 nm) limit their application in intravenous formulations, although they may be used to create biocompatible reservoirs.

The facile aggregation of SBA-15 particles caused by their size, which makes their tra fficking through alveoli and capillaries extremely di fficult, makes these materials ideal candidates for non-systemic therapies, for example, bone replacement and tissue regeneration. In our first contribution to the topic, we found that the osteostatin peptide, derived from parathyroid hormone-related protein (PTHrP107–111), could be e fficiently delivered using this kind of material [128]. A clear regenerative effect was observed, which permitted recovering bone mass in peri-implant bone regeneration in cavitary defects [129], as well as in osteoporotic subjects [130]. More recently, our investigations focused on the combination of this peptide with osteogenic elements embedded in bioactive glasses [142,143], finding an enhanced bone formation consequence of the exerted combination therapy.

In addition to these examples, other research groups also focused on the combination of osteoinductive peptides with bone-forming agents. Such is the case of the model reported by Mendes et al., which combined an osteogenic growth peptide (OGP, ALKRQGRTLYGFGG) with hydroxyapatite in an SBA-15-based formulation [131]. Therein, the authors found a substantial behavior di fference when hydroxyapatite was present, while deeper studies on the possibilities of such a combination were not addressed. Another interesting combination for remineralizing bone was reported by He and coworkers, who employed MCM-41 to prepare a nanosystem able to deliver a BMP-2-derived peptide (KIPKASSVPTELSAISTLYL) together with dexamethasone, a potent synthetic glucocorticoid [132]. Although, in this case, the peptide was not intended to be released, experimental results showed that its presence exerted a clear osteogenic induction. Moreover, the system was able to promote osteogenic differentiation on mesenchymal stem cells in vitro, as indicated by the upregulation of alkaline phosphatase (ALP) and other bone-forming proteins. Additionally, an increased calcium deposition was observed, demonstrating that mesoporous silica nanoparticles are adequate materials for bone engineering.

#### **5. Delivery of Nucleic Acids: Gene Modulation and Silencing**

Gene modulation and silencing are promising strategies to efficiently treat cancer, as well as cardiovascular or inflammatory-related diseases [20]. NAs are rapidly degraded by endonucleases and barely internalized by cells. Therefore, the biggest challenge today is to develop an effective gene delivery vector to transport and introduce NAs into the gene therapy target cells. These carriers must be easy to manipulate and show non-toxic properties in vitro and in vivo.

There are two main carriers for gene delivery therapy: viral and non-viral vector platforms [144]. For non-pathogenic viruses (retro and adenovirus), the most used platforms are viral vectors, but they present some limitations such as high cost and manipulation, immune response, or inadequate accessibility of genetic cargo internalized by cells [144]. Currently, research groups are focusing their efforts on the improvement of cheaper and easier preparation of non-viral platforms as cation polymers, liposomes, or inorganic porous nanoparticles such as MSNs [145]. In this respect, PEI is a widely used polycation in gene delivery systems due to its amino protonation improving gene delivery [20,144,145]. Liposomes are also commonly applied to enhance the cell internalization of NAs. Both polycations and liposomes present some limitations for therapeutic applications related to their molecular weight or disadvantageous gene escape and physicochemical instability, respectively [20,144,145]. Nevertheless, MSNs showed optimal properties for intracellular delivery of NAs due to their easy manipulation, high loading capacity into the mesopores, surface functionalization, and non-toxicity characteristics [20]. In addition, MSNs can be combined with polycations and other therapeutic drugs for personalized treatment related to cancer and other diseases.

In this regard, plasmid DNAs (pDNAs) are the most widely used gene therapy material to treat different diseases [20,144,145], although there is increasing interest in small interfering RNAs (siRNAs) and microRNAs (miRNAs) for therapeutic application. In this section, we focus on NA-loaded (DNA, siRNA, and miRNA) MSN-based drug delivery systems, as shown in Table 3 and Figure 5.





TnC: tenascin-C; COLL1A1: collagen type I; PMDFs: primary mouse dermal fibroblasts; HSCs: human stem cells; HDFs: human dermal fibroblasts; MEF: mouse embryonic fibroblastic

cells; rBMSCs: rat bone marrow stromal cells.

*Pharmaceutics* **2020**, *12*, 432

**Figure 5.** Types of nucleic acids and the possible loading strategies into MSNs for tuning gene expression on cells.
