*5.2. siRNAs*

Small interfering RNA (siRNA) are highly potent, short, double-stranded RNA molecules (20–25 bp) used in gene-based therapy of di fferent diseases that can be delivered in a sustained manner [178]. Moreover, siRNAs are anti-cancer targets because they can modify the expression of pro-apoptotic oncogenes and cell-cycle key regulators. However, siRNAs have a very short half-life and poor cell internalization capability [178]. In this regard, MSNs attracted pronounced consideration for the intracellular delivery of NAs due to their high loading capacity into the mesopores and surfaces. Polycations such as PEI combined onto the surface of MSNs permit a more stable encapsulation, loading, controlled release, and intracellular internalization of potential siRNA cargoes [178]. In addition, positively charged amine-rich nanosystems are required for a better result in siRNA/MSN cellular uptake rates [178]. Future in vivo efforts are necessary to validate the tissue-specific siRNA therapeutic efficacy using imaging techniques to analyze MSN biodistribution at the whole-body level. On the other hand, it is mandatory to find nanosystems able to e fficiently deliver those therapeutic siRNAs in vivo with enough e fficiency, as well as with protective features against nucleases, able to enhance their gene-silencing capabilities at the target tissues.

In this respect, Shen et al. [156] developed a universal siRNA multi-component system carrier based on MSNs functionalized with cyclodextrin-grafted polyethyleneimine (CP). CP o ffers a positive charge to improve the siRNA loading on MSNs through electrostatic interaction, allowing endosomal escape. To analyze the e ffectiveness of the proposed delivery system, an siRNA of the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2) was used, due to it being overexpressed

in several cancer types. Firstly, siRNA-loaded CP-MSNs were positively evaluated in vitro in MDA-MB-231 human breast cancer cells with higher rates of cellular internalization and gene silencing capability. The nanosystem showed specific tumor accumulation and gene silencing in an orthotopic mouse model of MDA-MB-231 cells, inhibiting tumor cell growth, invasion, and migration. Prabhakar et al. [157] designed a new nanosystem based on MSNs with expanded mesopores and pore–surface-hyperbranched PEI tethered with redox-cleavable organic linkers that could carry high concentrations of siRNAs. This stimulus-responsive nanosystem was highly internalized by MDA-MB-231 cells, releasing the cell-killing siRNA as cargo into the cytoplasm, followed by endosomal escape triggered by the intracellular redox situation, with high e fficiency in long-term gene knockdown for several days. In addition, long-term stability of therapeutic nanosystems is mandatory to provide personalized medicine. In this respect, lyophilization or freeze-drying are useful and common methods to ensure this aim. Ngamcherdtrakul et al. [158] successfully reported a lyophilized nanosystem consisting of MSNs modified with cationic polymers (PEI), PEG, and antibody (trastuzumab), which was loaded with a human epidermal growth factor receptor 2 (HER2) siRNA upon reconstitution. This nanosystem was successfully internalized by HER2<sup>+</sup> human breast cancer cells, BT474, decreasing their viability; it maintained a cake-like structure and retained hydrodynamic size, charge, siRNA loading ability, and silencing e fficacy. With the tested method, the lyophilized nanocarrier can be stored stably for eight weeks at 4 ◦C and at least six months at −20 ◦C.

Oral cancer is the seventh most frequent cancer and the ninth most frequent cause of death in the world [179]. About 90% of oral cancer is of squamous cell carcinoma type (SCC) [179]. Currently, surgery and radiation, combined or not with chemotherapy, are the main treatments, but this approach presents several limitations due to multidrug resistance; thus, new therapeutic drugs must be evaluated. Lio et al. [159] developed a topical formulation for the transdermal delivery of siRNA built on MSNs for the treatment of skin disorders. In this regard, transforming growth factor beta receptor I (TGFβR-1) is overexpressed in patients with skin SCC, and the anti-tumor e ffects of TGFβR-1 inhibitors were proven in several types of cancer. The nanosystem was built by loading TGFβR-1 siRNA into the pores of MSNs and further coating the resulting particle with a layer of poly-l-lysine (PLL) to improve transdermal delivery. The tested platform demonstrated positive cellular uptake rates and detection of specific gene biomarkers in human SCC cells in vitro. This system induced a topical delivery of TGFβR-1 siRNA targeting the SCC in a mouse xenograft model, decreasing the TGFβR-1 siRNA levels and reducing tumor volume. These results indicated that this platform is a promising non-invasive transdermal drug delivery system. Moreover, multidrug resistance plays an important role in the failure of oral cancer chemotherapy. In this sense, in a study by Wang et al. [160], a novel MSN loaded with both multidrug resistance protein 1 (MDR1) siRNA and DOX was evaluated in vitro and in vivo to directly kill tumor cells without the e ffect of multidrug resistance. MSNP-PEI@siRNA loaded with DOX was e fficiently transfected into human oral squamous carcinoma DOX-resistant (KBV) cells, decreasing the gene expression of MDR1 and increasing the apoptosis of these cells. This nanocarrier significantly reduced the tumor size and decelerated tumor growth rate in vivo, showing a huge potential therapeutic application for multidrug-resistant cancer. On the other hand, Shi et al. [161] developed hyaluronic acid (HA)-assembled MSNs nanosystems loaded with MDR1 siRNA and protein MutT homolog 1 (MTH1) inhibitor TH287. Downregulation of MTH1 protein expression increased oxidative stress levels and significantly decreased tumor survival and proliferation. TH287 is a potent small molecule that inhibits the MTH1 protein, and it could act as a potential chemotherapeutic drug. The HA-MSNs nanosystem was successfully internalized by CAL27 cancer cells, inducing a high rate of cell apoptosis when TH287 and MDR1 siRNA were present. In vivo, this nanocarrier reduced the tumor growth compared with untreated control and free TH287 in male Balb/c mice. These results sugges<sup>t</sup> that HA-MSN-based nanosystems are a promising vehicle to control the delivery of MDR1 siRNA and TH287, with a possible combination with other therapeutic agents for oral cancer treatment.

Regarding multidrug resistance as a persistent limitation for cancer treatment, Pan et al. [162] proposed a novel pH-responsive delivery nanosystem based on carboxylated MSNs (MSN-COOH) with a zeolitic imidazole framework-8 (ZIF-8) film synthesized for pore blocking and e fficient loading of anti-apoptotic B-cell lymphoma 2 (Bcl-2) siRNA. The nanosystem developed was e fficiently internalized by MCF-7/ADR and SKOV-3/ADR tumoral cells. The ZIF-8 film can be degraded in the acidic endo-lysosome and trigger the intracellular release of both siRNAs and potential antitumor drugs, decreasing cancer cell proliferation. In this regard, Choi et al. [163] developed a new biodegradable and biocompatible amine-free, neutral MSN-based nanocarrier for siRNA delivery. In this study, Bcl-2 siRNA was loaded via a calcium ion (Ca<sup>2</sup>+)-mediated interconnection between phosphates of siRNA and surface silicates of MSNs, reducing the risk of amine-related cytotoxicity and immunogenicity. This nanosystem showed positive results in internalization rates in tumoral SKOV-3 cells, inducing a significant decrease in cell viability when the siRNA was released, compared with MSNs or siRNA alone. This reduction in tumoral cell proliferation was dramatically enhanced when the nanocarrier was loaded with DOX. The therapeutic potential of the co-delivery system was also analyzed in SKOV3 xenografts in nude mice. The authors found that co-delivery of Bcl-2-siRNA and DOX MSNs-Ca2+ reduced tumor volume with an inhibition rate of 72%, higher than MSNs-Ca2+ loaded with DOX and MSNs-Ca2+ with siRNA. The results indicated that Ca2+-bound MSNs can act as an siRNA nanocarrier with a less toxic, more e ffective, and safer siRNA potential clinical application. On the other hand, patients with epithelial ovarian cancer (EOC) metastases relapse after initial e ffective chemotherapy treatment due to multiple drug resistance. In order to resolve this problem, Shahin et al. [164] proposed a novel nanoparticle delivery platform to reverse chemoresistance based on HA-MSNs loaded with TWIST siRNA. Hyaluronic acid (HA) is a ligand for CD44, which is overexpressed and correlates with worse prognosis in EOC. TWIST is a key protein implicated in epithelial–mesenchymal transition, cancer metastasis, angiogenesis, and drug resistance. The developed nanocarrier was successfully internalized in vitro by F2 and Ovcar8 cell lines, inducing a sustained TWIST gene knockdown. MSN-HA@siRNA showed safety and specific tumor targeting in an EOC mouse model. Mice treated with the complete nanosystem induced significant tumor reduction, and, when the authors combined this nanocarrier with cisplatin, the reduction of tumor growth was greater compared with the corresponding control groups.

For the metastatic spread of cancer, growth of the vascular network is a critical event [29]. Vascular endothelial growth factor (VEGF) is a glycoprotein and key mediator of angiogenesis in tumoral cells [180]. Keeping this in mind, Zheng et al. [165] proposed a new nanocarrier based on MSNs functionalized with FA-targeted tumoral cells and co-loading of VEGF siRNA and ursolic acid (UA), a pentacyclic triterpenoid with anticancer e fficacy. HeLa (FA receptor-overexpressed) and HepG2 tumoral cell lines were used to analyze the MSN-FA-UA@siRNA nanosystems in vitro. The MSN-FA nanocarrier enhanced transfection e fficiency and siVEGF stability, improving the targeted antitumoral efficacy of siVEGF and UA, with a significant decrease in cancer-related VEGF protein levels in HeLa cells. In this sense, the same authors [166] developed an alternative MSN nanosystem consisting of an asialoglycoprotein receptor (ASGPR)-targeting drug delivery system for co-delivery of siVEGF and sorafenib (SO), a multi-kinase inhibitor that can target VEGF for the inhibition of tumor growth and neo-angiogenesis in hepatocellular carcinoma (HCC). Lactobionic acid (LA) is derived from lactose oxidation, and it was able to e ffectively target human HCC. In addition, ASGPR is overexpressed in several types of cancer cells, facilitating hepatic infection, and it is specifically recognized by glycoproteins which bind with it. The MSN-LA-SO@siVEGF nanocarrier induced synthesis (S) phase cell-cycle arrest, increasing the anti-cancer e fficacy (cell apoptosis) of SO and siVEGF through the active targeting of LA in ASGPR-overexpressing Huh7 tumoral cells. Furthermore, this nanosystem enhanced the siVEGF transfection e fficiency and inhibited the expression of angiogenesis-related VEGF proteins in these tumoral cells. Taking both studies together, these types of functionalized MSN-based nanosystems could be potential nanocarriers for targeted delivery of anti-tumoral drugs and VEGF siRNAs to improve anti-tumor capability.

On the other hand, fibrotic diseases are related to augmented oxidative stress and upregulation of pro-fibrotic genes [181]. Oxygen species (ROS), nicotinamide adenine dinucleotide phosphate

(NADPH) oxidase 4 (NOX4) and heat-shock protein 47 (HSP47) are implicated in this disease [181], producing disproportionate collagen synthesis. Morry et al. [167] proposed an MSN platform with PEI and PEG coating that effectively delivered HSP47 siRNA in vitro and in vivo. This nanocarrier induced a knockdown of the HSP47 gene and a decrease in ROS and NOX4 levels in an in vitro model of fibrosis based on TGF-β-stimulated fibroblasts. Intradermal administration of MSNs@siHSP47 successfully decreased HSP47 protein levels in a bleomycin-induced scleroderma mouse model in vivo, reducing ROS production and different pro-fibrotic markers. These nanocarriers could be a very interesting platform to treat both fibrotic and inflammation diseases. On the other hand, it is necessary to develop anti-fibrotic drugs for chronic liver fibrotic diseases. Hepatic stellate cell (HSC) activation is present in the progression of liver fibrosis, secreting extracellular matrix proteins as tenascin-C (TnC). Vivero-Escoto et al. [168] proposed an MSN@siTnC based nanosystem as a favorable alternative to evaluate the siRNA therapy of chronic liver disease in preclinical trials. This novel nanocarrier was successfully internalized by human stem cells in vitro and produced a downregulation in TnC mRNA and protein levels, inducing a decrease in inflammatory cytokine expression by macrophages and hepatoma migration. These promising results must be tested in a liver fibrosis in vivo model.

As demonstrated in this section, siRNA-MSN based nanosystems were widely studied and showed a high efficiency in silencing genes and reducing tumoral growth related to cancer processes in vivo. Nevertheless, in tissue engineering applications, scaffold-mediated delivery is desired to achieve local and sustained release of drugs. In this respect, Pinese et al. [169] developed an MSN-PEI@siRNA nanocarrier delivered from electrospun scaffolds via surface adsorption and nanofiber encapsulation. These nanosystems were either combined with the surfaces of nanofiber substrates or directly encapsulated. MSN-PEI@siRNA coated scaffolds offered sustained availability of siRNA for at least 30 days and five months in the case of MSN-PEI@siRNA-encapsulated scaffolds within the electrospun fibers in human dermal fibroblasts cells in vitro. The scaffolds showed excellent bifunctionality in vitro and in vivo by targeting collagen type I (COLL1A1) as a target gene to reduce fibrous capsule formation. These platforms were subcutaneously implanted in a rat model in vivo and COLL1A1 siRNA/MSN-PEI induced a reduction of 45.8% in fibrous capsule formation after four weeks compared to negative siRNA treatment. This method improved siRNA internalization rates and sustained targeted protein silencing in vitro and in vivo. The nanoplatform proposed by these authors can be used to release and internalize siRNA/miRNA combined with other specific low-molecular-weight drugs in long-term tissue engineering applications and cancer. In addition, the current pharmacological therapy of bone diseases such as osteoporosis presents several limitations related to bioavailability and toxicity. In this sense, gene knockdown through siRNA delivery received grea<sup>t</sup> attention as a promising treatment in osteoporosis [182]. In a recent study by Mora-Raimundo et al. [170], a nanosystem based on MSN-PEI, which can effectively deliver SOST siRNA and osteostatin inside cells, was evaluated in vitro and in vivo. The SOST gene inhibits the Wnt signaling pathway, decreasing osteoblast differentiation, whereas osteostatin is a pentapeptide with osteogenic and anti-osteoclastic properties [183]. SOST siRNA/MSN-PEI loaded with osteostatin induced a dramatic suppression of SOST gene expression in mouse embryonic fibroblastic cells with the consequent upregulation of several osteogenic markers with a synergistic effect. In vivo, the complete nanosystem was injected in the femoral bone marrow of ovariectomized mice, and the results were in concordance with in vitro studies, knocking down SOST and upregulating the osteogenic marker levels in a synergistic manner. The nanoplatform proposed by these authors was able to transport, co-deliver, and internalize SOST siRNA and osteostatin in cells, preserving its activity and achieving an effective silencing effect.
