*5.3. miRNAs*

MiRNAs are small endogenous noncoding RNA molecules (17–25 nucleotides) that act as regulators of gene expression and downregulate target proteins via miRNA degradation or translational inhibition [184]. The aberrant expression of miRNAs in several cancers is associated with several mechanisms such as cellular differentiation, proliferation, survival, and apoptosis [184,185]. MiRNAs can accomplish a gene knockdown e ffect by regulating multiple miRNAs compared to siRNA, which is very useful when treating complex multifactorial diseases such as cancer [184,185]. Several drugs based on miRNAs were used in clinical trials in cancer [186]; however, the e ffective and safe delivery of anti-miRNAs or miRNA mimics is a key challenge for clinical applications.

Regarding these objectives, Li et al. [171] proposed a novel nanosystem based on MSN functionalized with polymerized dopamine (PDA) and AS1411 aptamer loaded with microRNA-155 (anti-miRNA-loaded MSN@PDA-Apt) for the specific treatment of colorectal cancer (CRC). Upregulation of miR-155 (oncogenic microRNA) was found in several cancer-related pathways, including in CRC. Firstly, the authors demonstrated that nuclear factor kappa B (NF-κB), an important transcription factor with a crucial role in the process of tumor growth, has a positive feedback loop with miR-155 in vitro and in CRC tissues. MSNs-anti-miR-155@PDA-Apt decreased miR-155 expression in SW480 cells and successfully targeted the CRC tumor, leading to gene knockdown and tumor growth reduction, thanks to both active targeting of AS1411 aptamer and passive targeting of the EPR e ffect. In addition, the same authors [172] explored the miR-328 pathway and developed a new pH-responsive nanoplatform consisting of MSNs-miRNA-328 decorated with PDA, a cell adhesion molecule aptamer, and bevacizumab (MSNs-miR-328@PDA-PEG-Apt-Bev) for the dual-targeting treatment of CRC. MiR-328 is a tumor suppressor downregulated in several human cancers, including CRC, and it is correlated with drug resistance. The authors demonstrated for the first time that CPTP, a ubiquitously expressed lipid transfer protein associated with inflammation and CRC, is a direct target of miR-328. MSNs-miR-328@PDA-PEG-Apt-Bev increased miR-328 levels and inhibited CPTP expression in SW480 cells, increasing binding ability and showing much higher cytotoxicity in vitro and in vivo. In addition, this nanoplatform showed e fficient gene silencing and tumor growth inhibition of the target tumor in a SW480 xenograft mouse tumor model in vivo. Taken together, these results indicate that the MSN-based functionalized nanoplatforms reported herein show promise for miRNA therapy in cancer, specifically CRC. In a recent study by Ahir et al. [173], a nanosystem based on MSN-HA@miRNA with an appended PEG–poly(lactic-*co*-glycolic acid) (PLGA) polymer to target triple-negative breast cancer (TNBC) was evaluated in vitro and in vivo. In this context, miR-34a is downregulated and miR-10b is upregulated in TNBC disease, inducing tumorigenesis and metastatic dissemination. These authors proposed the MSN nanocarrier for co-delivery of miR-34a-mimic and antisense-miR-10b, targeting the CD44 receptor. In vitro, this nanosystem showed positive results in terms of cellular internalization rates, release profile, and a subsequent pro-apoptosis e ffect in human mammary carcinoma cell lines (MDAMB-231 and MDAMB-468). In vivo studies exhibited a high specificity in TNBC tumor targeting, leading to e ffective tumor growth reduction and metastasis delay in mice.

As previously mentioned, MSN nanosystems emerged as a promising bone regeneration methodology in bone tissue engineering. In this regard, Yan et al. [174] developed a novel miR-26a delivery nanosystem based on MSNs. MiR-26a was confirmed to regulate several pathways of osteogenic di fferentiation and promote bone regeneration. The authors demonstrated the protection effectiveness of the vectors to the miRNA and the positive internalization rates in rat bone marrow stromal cells (BMSCs) in vitro, releasing miR-26a into the cytoplasm without cytotoxicity e ffects. This nanosystem promoted stem-cell osteogenic di fferentiation, inducing an increase in alkaline phosphatase activity and mineralization, as well as in several genes and proteins implicated in osteogenesis in BMSCs. The nanocarrier proposed by these authors provides new methods and strategies for the delivery of mRNAs in bone tissue engineering, but it is necessary to further validate them in vivo.

On the other hand, a combined siRNA/miRNA therapy would be a very interesting approach, especially for cancer treatment, targeting multiple disease-related pathways and silencing specific genes. Currently, the use of this combination in clinical studies shows several limitations such as the availability of safe and e ffective systemic delivery nanocarriers with e fficient tumor penetration. In this respect, Wang et al. [175] developed multifunctional tumor-penetrating MSNs for co-delivery of siRNA (siPlk1) and miRNA (miR-200c), upregulated in several types of tumor and implicated in cancer development and progression. In addition, the authors functionalized the nanosystem to facilitate endosomal scape using a photosensitizer indocyanine green (ICG) and surface conjugation of the iRGD peptide to allow deep tumor penetration. The complete nanosystem induced an increase in internalization rates in MDA-MB-231 cells and three-dimensional (3D) tumor spheroids in vitro, whereas ROS produced by ICG upon light irradiation induced the endosomal scape of the siRNA/miRNA into the cytoplasm with a deleterious e ffect on cell viability. In vivo, a significant reduction in tumor growth and metastasis upon short-light irradiation was found when the nanosystem was intravenously administrated in mice with metastatic breast cancer.

## **6. Delivery of Glycan-Based Biomolecules**

Beyond the use of proteins, peptides, and nucleotides, glycan-based structures were also successfully employed in MSN-based delivery. Among them, the most recurrent examples are reported with heparin, chondroitin sulfate (CS), chitosan, and HA. Those compounds, apart from being important components of structural matrices, are known to a ffect many biochemical processes. For instance, HA is a valuable targeting element for the development of new therapeutic tools, as it is able to interact with CD44 and CD168, which are upregulated in many cancerous cells and are closely related to cell adhesion and migration in metastases [187,188].

Similarly, chitosan [188–190] and chondroitin sulfate [191–193] were employed for the development of high colloidally stable and biocompatible nanosystems with certain targeting capabilities. Moreover, as both substances are important structural components of extracellular matrices on vertebrates and crustaceans, they can act as bioactive components for tissue regeneration. Such was the case in the contributions by Kavya et al. [194] and Porgham Daryasari et al. [195], who employed those elements to induce osteogenesis through two di fferent approaches. In the first approach, Kavya et al. designed a crosslinked chitosan/CS sca ffold reinforced with nanometric SiO2. In this system, the presence of silica permitted improving the mechanical properties and obtaining slower degradation, while the glycans permitted obtaining an adequate moisture swelling for creating a favorable environment for cell proliferation [194]. In the article by Porgham Daryasari et al., the authors employed chitosan-coated, dexamethasone-loaded MSNs as a bioactive component in a poly-l-lactic acid sca ffold. The system was proven to induce osteogenic di fferentiation of human adipose stem cells according to the ALP activity [195]. Although both models o ffered promising results, the absence of osteoregeneration experiments in vivo makes these materials a mere design exercise, in which the presence of glycans is not completely justified. Apart from its use as a stabilizer, CS was also successfully employed as a functional coating able to interact with blood lipoproteins through a double e ffect: electrostatic interaction plus saccharide recognition [196]. Although these CS-modified dendritic MSNs permitted selectively binding and isolating low-density lipoproteins (LDL) for analytical purposes, the potential of such a nanosystem as a therapeutic agen<sup>t</sup> is of enormous interest, as it may enable an interesting therapeutic e ffect against hypercholesterolemia and blood vessel atheromatosis.

Heparin is a highly sulfated, negatively charged glycosaminoglycan with outstanding properties as an anticoagulant. Beyond injectable applications, heparin is employed as an anticoagulant surface in medical devices like test tubes and dialysis machines. Regarding additional uses in combination with mesoporous silica, its capacity as a targeting moiety was also described against human hepatocyte carcinoma HepG2 [197,198] and HUVEC cell lines [198].

Additionally, as a consequence of its anticoagulant e ffect, some investigations focused on improving its release profile from silica-based carriers as nanodepots. Along this line, the research articles authored by Zhu and coworkers performed two systematic studies on how di fferent types of mesoporous silica, with varying pore sizes and functionalization, a ffected the loading-release process [199,200]. Based on their results, the authors established that a slight pore enlargement of MCM-41 yielded better adsorption and release. Indeed, they also demonstrated that the presence of amino groups in the mesopores increased the amount of heparin adsorbed, albeit at the expense of slowing down the release [199]. With regard to SBA-15, they found that, apart from pore size and charge, the presence

of rough pore surfaces caused by carbonaceous deposits upon calcination boosted both the retention and the release of heparin, improving the performance of such a material and enabling access to better-performing nanodepots.

Apart from its use as a drug delivery agent, there are examples of heparin-loaded silica-based materials used as anticoagulant coatings for medical devices. For example, Wei et al. reported the use of SBA-15 to prepare a heparin-releasing anticoagulant coating [201]. Their system employed amino-modified mesopores to favor heparin loading and delay its release. To incorporate these particles into chosen substrates (silicon wafer, glass, or polyvinylchloride), the authors coated the particles with polydopamine and the substrate with a catechol-modified chitosan polymer. This configuration permitted depositing the negatively charged, heparin loaded MSNs onto chitosan, which was further bound to the catechol–dopamine coating of substrates. Finally, to prevent undesired detachment, this dopamine–catechol bond was oxidized with NaIO4 in order to create a non-degradable polymeric layer. When the authors tested this coating against blood, they found a very low fibrinogen adsorption, as well as platelet adhesion and hemolysis, which is in concordance with a sustained anticoagulant release and good biocompatibility. More recently, Wu et al. reported a similar approach but with an added feature: antibacterial adhesion. To achieving this, the authors loaded the mesopores of MSNs with two glycan-based structures: agarose for an antibacterial e ffect and heparin as an anticoagulant [202]. The agarose-heparin loaded MCM-41 nanoparticles were electrostatically immobilized onto the prepared device, i.e., an amino-modified silicone film. Therefore, the final coating presented a negative charge and exposed mesopores, which were able to produce e ffective agarose and heparin release. The system demonstrated a low hemolytic e ffect comparable with that of the previous model but with an additional antibacterial e ffect against *E. coli*, gaining additional applicability. However, regarding the design, the possibility of detaching SBA-15 particles within the bloodstream has to be considered as a potential risk and must be accounted for in the further development of such devices.

## **7. Challenges and Future Perspectives**

As reviewed, the number of developing therapeutic hybrid nanosystems employing biomolecules together with mesoporous silica nanoparticles is enormous, as reported over the last few years. In general, and according to the literature here visited, cancer therapy is by far the most active research field and the one that exploits the most up-to-date technological advances such as PEGylation, controlled drug delivery, or the implementation of hybrid nanomaterials, among many others. However, such technology permitted developing alternative therapies to cancer, applied to other not-so-common diseases with a promising prognosis. This fact is largely due to the particular structure of MSNs, with a high capacity to load and release therapeutic molecules within their porous matrix. However, the advancement of knowledge and the need to adapt these nanomaterials to advanced preclinical in vivo models, with new aspects in loading and release systems, must be considered when designing these medical nanosystems. In this context, short therapeutic peptides and small molecules seem to be the ideal target for transfer to preclinical studies because they can be easily designed, developed, functionalized, and coupled. Among these new emerging therapeutic strategies with biomolecules that came to light recently, the most promising for implementation together with mesoporous silica materials seem to be immune therapy, gene modulation, and anti-infective nano-agents, as can be determined from the number and quality of related papers. In any case, there are di fferent issues to be addressed regarding the nanocarriers themselves such as the absence of reliable in vivo pre-clinical studies, methods of clearance of the nanoplatforms, and how di fferent particles properties (shape, size, pore symmetry and connectivity, and chemical composition) a ffect their application in nanomedicine.

**Author Contributions:** D.L. and R.R.C. contributed equally to this work, M.V.-R. coordinated the work. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors want to acknowledge financial support from the European Research Council ERC-2015-AdG-694160. R.R.C especially acknowledges Centro de Investigación Biomédica en Red for a postdoctoral contract.

**Conflicts of Interest:** The authors declare no conflicts of interest.
