*5.1. DNA*

As we previously mentioned, MSNs can be functionalized with PEIs for better DNA molecule adsorption and, thus, for a more effective intracellular delivery, protecting NAs from endonuclease degradation due to their positive charge. These positive charges interact with negatively charged cell membranes, inducing higher cell internalization rates and cell death. For example, Zarei et al. [146] developed a functionalized nanosystem based on phosphonate MSNs-PEI loaded with a lysosomotropic factor, chloroquine (CQ), complexed with plasmid DNA. The resulting cell internalization and viability of this pDNA-MSN-PEI was analyzed with the fluorescent protein plasmid (pGFP (green fluorescent protein)), showing a significant increase in transfection of pGFP into the mouse neuroblastoma cell line Neuro-2A. In this respect, Xia et al. [147] proposed the use of a cationic MSN-PEI nanocarrier with a potential therapeutic application. The resulting cationic surface of MSNs facilitated the DNA attachment in order to balance the cationic 10-kDa PEI and, hence, achieve nontoxic effects and higher cellular uptake rates. This nanosystem was loaded with a plasmid DNA (pEGFP) and siRNA construct that was capable of knocking down GFP expression in hepatocellular carcinoma mouse (HEPA-1) cells, with a fluorescent GFP expression of 70% in these cells. In addition, the authors loaded the cationic MSN-PEI nanocarrier with paclitaxel, a hydrophobic anti-tumoral drug used in pancreatic tumor treatment, increasing the cell internalization and delivery of this drug in HEPA-1 cells. On the other hand, Song et al. [29] demonstrated that controlling the nanotopography of MSNs as pDNA vectors had an influence on the transfection efficacy. For example, ambutan-like MSNs-PEI with spiky surfaces showed pDNA-binding capability and a transfection efficacy of 88% in HEK-293T cells, which was a higher rate compared with other MSNs systems. In addition, the authors demonstrated that these types of surface spikes of MSNs induced a continuous open space with two objectives: binding DNA chains through multivalent interactions and protecting the gene cargo covered in the spiky layer against enzymatic degradation, without negative effects on transfection rates. These results indicated that this nanosystem is an interesting approach as a non-viral vector for effective gene delivery.

In a study by Wang et al. [148], an MSN based nanoplatform functionalized with a DNA gate was evaluated in vitro. In this report, the authors developed a fluorometric method for adenosine triphosphate (ATP) recognition using rolling circle amplification (RCA) consisting of proximity ligation-mediated amplification. In addition, the nanosystem was functionalized with graphene oxide modified with folic acid (FA) as a DNA vehicle and loaded with DOX, to evaluate the control release efficacy in HeLa tumoral cells. Following the RCA process, long DNA chains that contained a complementary strand to the DNA at the gate permitted dehybridizing such a nanogate and producing DOX release into tumoral cells. This nanoplatform was effectively internalized by the FA receptor, upregulated in those tumoral cells, while the DOX release induced increased cell toxicity compared with control systems, improving the targeted cancer therapeutics on these HeLa cells. In another study by Wang et al. [149], a DNA-capped MSN nanocarrier loaded with DOX for tumor marker-triggered on-demand drug release was developed and evaluated in vitro. As a first step, a DNA biomolecular gate adsorbed on the MSN–NH2 was developed at neutral pH via electrostatic adsorption. In the absence of a stimulus, the pores were locked, and it was not possible to release the drug. However, when the nanosystem recognized the internal stimulus (survivin messenger RNA (mRNA) or miR-21, overexpressed biomarkers in cancer), the DNA caps were removed from the MSNs, allowing DOX release. This stimulus-responsive behavior was confirmed by different microscopy techniques in cell-acute myeloblastic leukemia (HL-60) cells. This nanocarrier was effectively internalized by these cells, releasing DOX cargo into the cytoplasm and inducing significant cell death. Therefore, this nanoplatform is a very useful novel system for both imaging diagnosis and therapeutic controlled drug delivery applications. In this regard, Li et al. [150] designed and evaluated another anti-tumoral and gene drug co-delivery nanosystem with on-demand release properties in vitro and in vivo. For this purpose, the authors developed dendritic MSNs modified with imidazole groups employing a Schiff-base imine linkage, which permitted loading DOX within the pores and electrostatically depositing the survivin short hairpin RNA (shRNA)-expressing plasmid (iSur-pDNA). The imidazole functionalization triggered efficient endosomal escape, improving the accumulation of iSur-pDNA and gene knockdown efficacy. Such nanosystems were successfully internalized by QGY-7703 hepatoma cells and decreased tumoral cell viability in vitro, due to the pH-sensitive co-delivery of DOX and iSur-pDNA. In addition, this nanoplatform induced tumor growth reduction in H-22 tumor-bearing mice, indicating that it could be a promising nanocarrier for co-delivery cancer treatment.

On the other hand, aptamers are short single-stranded oligonucleotides with high affinity and specificity to several molecule targets, and they exhibit potential properties as therapeutic and diagnostic factors in different diseases [176]. In this regard, Sun et al. [151] developed an MSN-based nanocarrier with different DNA molecules on the surface for self-assembly. For the controlled delivery of anti-tumoral drug cargo (DOX), the authors used an aptamer oligonucleotide as a gatekeeper and other oligonucleotides on the MSN surface to allow DNA-guided immobilization bearing single-stranded capture oligonucleotides. This nanovalve induced an increase in cell adhesion rates in MCF-7 adenocarcinoma cells, with an efficient triggered release of DOX drug in these cells. The DNA-directed self-assembly aptamer-based nanosystem is efficient in surface-bound monolayers and can be used as a delivery system for different applications and treatments, allowing site-specific delivery of anti-tumoral drugs. In the same way, Li et al. [152] developed a dual multi-locked DNA valve HMSN-based nanosystem to intracellular cancer-related mRNAs for controlled DOX drug release. The upregulated endogenous targeted mRNAs (K1 and GalNAc-T mRNAs) were able to dehybridize the multi-locked DNA valves and open the pores to release DOX. As a result, a synergic effect was obtained, caused by the intratumoral delivery of the drug together with the depletion of TK1 mRNA, implicated in cell division as a tumor growth factor, and GalNAc-T, upregulated in several tumoral cells. The nanocarrier was evaluated in vitro in different cancer cells, inducing higher rates of cytotoxicity compared to non-tumoral cells. In an in vivo study, Pascual et al. [153] designed another novel non-viral drug delivery and diagnostic nanocarrier based on a capped MSN-NH2 hybrid platform, which was

loaded with DOX and gated with Mucin 1 (MUC1) aptamer (S-apMUC1). MUC1 is a protein present in the cell surface and upregulated in breast cancer cells. The S-apMUC1-MSNs nanosystem was efficiently and specifically internalized only by tumoral MDA-MB-231 cells related to MUC1 receptor overexpression of these cells. After S-apMUC1-MSNs internalization by tumoral cells, the DOX cargo was released into the cytoplasm of tumoral cells, inducing a decrease in cell viability. This nanoplatform exhibited reduced cargo release when DNAse I was not present. In vivo, S-apMUC1-MSNs were radionuclide-labeled with 99Tc for a radio-imaging study in MDA-MB-231 tumor-bearing Balb/c mice. S1-apMUC1-Tc displayed significant tumor signaling and accumulation in these mice. These results sugges<sup>t</sup> that MSN nanosystems capped with aptamers showing radiopharmaceutical properties are promising hybrid nanomaterials in the clinical context.

Of particular interest is a study by Srivastava et al. [154], where the authors proposed a novel telomerase-responsive delivery of DOX loaded in an MSN nanosystem against telomerase-positive tumoral cells. DOX loading was performed using a telomeric repeat complementary sequence and a telomerase substrate primer sequence, allowing controlled release in MCF-7 cancer cells. This functionalized nanocarrier was e fficiently internalized, and it significantly reduced the tumoral cell viability, with an inhibition of function when a specific telomerase inhibitor was present. This oligo-wrapped nanoprobe specific for telomerase was used in a telomerase-positive Dalton's lymphoma mice model. The nanosystem induced a significant inhibition of tumors, enhanced survival, and re-established histopathological parameters, including neo-angiogenesis.

Furthermore, DNA vaccination was proven to provide an immune response to viruses or infectious diseases [177]. In this regard, Wang et al. [155] developed a layered double hydroxide (LDH) MSN nanocarrier as a vaccine delivery platform and immune stimulant, using a GFP expression plasmid as model DNA. The pDNA-MSN-LDH nanosystem showed high internalization rates in monocyte-derived dendritic cells (MDDCs) and stimulated macrophage activation via nuclear factor kappa B (NF-κB) signaling pathway, increasing IFN-γ, IL-6, CD86, and major histocompatibility complex class I (MHC-I). In vivo, the nanocarrier immunized BALB/c mice, indicating that the DNA vaccine-MSN-LDH system increased the serum antibody response and promoted T-cell proliferation, skewing T helper to Th1 polarization. Taken together, these results indicated that MSN-LDH nanosystems could operate as a potential non-viral gene delivery platform.
