**1. Introduction**

Small interfering RNA siRNA suppress expression of genes by targeting the mRNA expression. Targeted delivery of siRNA to specific cells is highly desirable for safe and efficient RNAi-based therapeutics [1]. However, the half-life of nucleic acids in the bloodstream is short due to the degradation by endo or exonucleases and rapid clearance [2]. One strategy to solve this challenge is developing siRNA delivery systems. Nanoparticles can be defined as particles less than 100 nm in diameter, these systems can be composed by different materials and are employed according to their purpose [3,4]. For this area, the most widely used systems are polymeric particles, nanoemulsions, nanocrystals, solid lipid nanoparticles, and liposomes [5]. The organic particles used for drug delivery application are micelles, liposomes, polymers, dendrimers, and nanogels. They have versatile surface building blocks for efficient endocytosis and loading [6]. There are numerous advantages to using nanoparticles: (I) Increased bioavailability, (II) dose proportionality, (III) decreased toxicity, (IV) smaller dosage form, (V) stability of drugs dosage forms, and (VI) increased active agent surface area resulting in a faster dissolution [7]. Ideally, nanoparticles should be stable in circulation to protect and deliver their therapeutic load (drug) into recipient tissue; have good penetration and retention in the target tissue so that drug release occurs within the therapeutic window; and ultimately be organically excreted to avoid long term accumulation toxicity [8]. Approaches to drug targeting and delivery may be facilitated by the enhanced permeability and retention (EPR) effect. This effect occurs due to the large

endothelial tissue fenestrations which are characteristic of the rapid growth of tumor blood vessels. Therefore, the nanoparticles passively diffuse through the microenvironment targeting the tumor tissues [8].

Although nanocarrier technology has improved, its lack of target specificity limits its widespread use, to overcome this issue and address the lack of specificity is the generation of functionalized nanoparticles, i.e., second generation nanoparticles [8]. Nanoparticle surface functionalization occurs through the fixation of a ligand that interacts with specific tissue-specific receptors, to optimize the administration of the target, selectively transporting it to the binding site [9]. One of the advantages of taking drugs directly to specific tissues is the ability to use relatively more toxic and efficient drugs with less risk of collateral damage to other body tissues. In the case of cancer, drugs could be targeted at tumors, avoiding the systemic side effects of traditional therapies. The functionalization includes surface conjugation of chemicals or bio molecules, like folic acid, biotin molecules, peptides, antibodies, aptamers, short, single stranded RNA or DNA oligonucleotides, proteins, and oligosaccharides, to enhance the properties and hit the target with high precision [10]. In order to provide targetability, aptamers have been widely used due to (I) their capacity of binding to target proteins with a high affinity and specificity, (II) having already been shown to have antibody-like characteristics, and (III) the fact that they are relatively smaller and less immunogenic. All of these useful properties make aptamers attractive in therapeutic and diagnostic fields [11].

#### **2. Aptamer in the Delivery of Therapeutic Nanoparticles Containing siRNA, shRNA, and miRNA**

The origin of siRNA nanoparticles targeted delivery through aptamers dates to 1998. Guo et al. treated T cells with an RNA nanoparticle consisting of a dimer of the packaging RNA (pRNA) derived from the DNA-packaging motor of bacteriophage phi29 loaded with a siRNA for survivin mRNA and conjugated with a CD4 specific aptamer [12]. Hu et al. used the same platform to create a nanoparticle containing an siRNA for ICAM 1 conjugated with aptamer FB4 directed against the mouse transferrin receptor. The in vitro results showed a decreased of ICAM-1 expression and blocked the adhesion of monocytes [13].

From this date, many types of platforms and supports were developed so that siRNA could be targeted through aptamers. To target cells using aptamers, Afonin et al. designed multifunctional siRNA nanoparticles aiming at the silence of multiple HIV-1 genes [14]. To show the feasibility of siRNA delivery using nanorings, Li et al. developed a nanorings construct functionalized with J18 RNA aptamers specific for the human epidermal growth factor receptor (EGFR) [15]. The nanoring design can achieve cell-targeting properties, but the internalization and functional effects of siRNAs bound to aptamer-nanorings were not studied [16]. Zhao et al. developed a nanoparticle to treat anaplastic large cell lymphoma (ALCL). The nanocarrier incorporated both anaplastic lymphoma kinase (ALK) siRNA and a CD30 aptamer onto nano-polyethyleneimine-citrate leading to growth arrest and apoptosis in vitro [17]. Powell et al. tested the possibility of conjugating an aptamer A6 which can bind to human epidermal growth factor receptor 2 (HER-2) receptors on breast cancer cells to P-gp siRNA siRNA-containing nanoparticles. The knockdown of P-gp was increased significantly, decreasing a resistance to chemotherapeutics in the cells [18]. To overcome multi-drug resistance (MDR), an MD- specific aptamer-conjugated grapefruit-derived nanovectors (GNVs) was used for co-delivery of siRNA and doxorubicin (DOX), resulting in a potent anti-tumor activity [19].

Stable nucleic acid lipid particles (SNALPs) also used to deliver siRNA through aptamers. In this study, the authors coupled the transferrin receptor aptamer in SNALP loaded with siRNA. The aptamer-targeting enhanced siRNA uptake and target gene knockdown in cells [20]. Another aptamer–liposome–siRNA delivery system was developed by Alshaer et al. In this model, siRNA complexed with protamine and aptamer anti-CD44, a cell surface biomarker overexpressed in many tumors, was used as targeting delivery for the first time [21]. They confirmed the silencing disease-related genes in tumors [18].

To target prostate cancer cells, Kim et al. used shRNAs against the gene BCL-xL and the PSMA (prostate-specific membrane antigens) aptamer (A10–3) conjugated to a polyethyleneimine (PEI)-PEG construction. In order to amplify the therapeutic response and use different drugs in synergy, the authors also included DOX in aptamer-functionalized nanoparticle, and this approach led to the death of the cancer cells, demonstrating the effectiveness of the combination therapy [22]. Likewise, the PSMA aptamer was used to target delivery of miRNA (miR-15a and miR-16–1) identified as tumor suppressor genes in prostate cancer. The nanoparticle based on polyamidoamine and PEG allowed selective killing of prostate cancer cells [23]. Xu et al. used a multifunctional envelope-type nanoparticle platform constituted of two polymers that co-incorporate siRNA. For the nanoparticle functionalization was used *S*,*S*-2-(3-(5-amino-1-carboxypentyl)ureido)pentanedioic acid (ACUPA) ligand that can specifically bind to PSMA. This study reached an efficient gene silencing in prostate cancer tumor cells in vivo [24].

Lv et al. used Polyamidoamine (PAMAM) dendrimers functionalized with EGFR aptamers to co-deliver shRNA to survivin and erlotinib. Survivin shRNA is used to activate apoptosis while erlotinib is a tyrosine kinase inhibitor. This therapeutic approach in association with Chloroquine acted synergistically to reverse erlotinib resistance in EGFR mutation-positive of non-small-cell-lung cancer (NSCLC) [25].

Another strategy is use of siRNA-aptamer chimera to compose the nanoparticle, and the siRNA-aptamer chimera has emerged as a promising approach for efficient delivery of siRNA to specific cell types, owing to its low immunogenicity, ease of chemical synthesis and modification, and the outstanding targeting specificity of the aptamer [26]. However, in some cases, endosome escape has been reported. To avoid this problem, it has been used siRNA-aptamer chimeras in nanocarriers. Bagalkot and Gao developed a new technology for linking siRNA-aptamer chimeras to carrier nanoparticles [27]. In this new technology, the siRNA block should become the anchor point for interaction with cationic nanoparticles for reduced enzymatic degradation and nonspecific interaction with cells and tissues, whereas the aptamer block should stay on the outside with minimized interaction with the nanoparticle surface. This approach opened new opportunities in targeted delivery based on siRNA-aptamer chimeras.

In recent study, to evaluate cell viability and genes expression in metastatic breast cancer cells, Jafari et al. designed chitosan nanoparticles for co-delivery of Docetaxel and insulin-like growth factor receptor 1 (IGF-1R) siRNA. Docetaxel is a cytotoxic anti-cancer drug approved for the treatment of metastatic breast cancer. IGF-1R signaling is important in tumor growth, development, and its metastasis. The MUC1 aptamer was conjugated to a chitosan based nanocarrier. MUC1-aptamers have been used to targeted drug delivery to several types of cancers, including breast cancer cells [28]. Another study in the cancer field shows an efficiency of EGFR aptamer-conjugated liposome loaded with special AT-rich sequence binding protein 1 (SATB1) siRNA. The genome organizer (SATB1) expression decreased in vitro and in vivo studies with a consequent decrease in choriocarcinoma [29]. A novel targeted delivery platform to mediated gene-silencing in cancer cells was described by Ayatollahi et al.; the 10-bromodecanoic acid (10C) and 10C-PEG was loaded with shRNA plasmid for specific knockdown of BCL-xL protein and was conjugated to AS1411 aptamer for targeting to nucleolin on cancer cells [30]. A synergistic cancer cell death was achieved using a (AS1411) nucleolin aptamer-carbon nanotubes (CNTs) as biological carriers containing BCL-xL-specific shRNA and DOX [31].

Recently, a new types of siRNA delivery by aptamers have been demonstrated. Xu et al. used the three-way-junction (3WJ) RNA to couple epithelial cell adhesion molecule (EpCAM) aptamers aiming to deliver Delta-5-Desaturase (D5D) siRNA. This enzyme acts on dihomo-γ-linolenic acid DGLA, preventing the production of 8-hydroxyloctanoic acid (8-HOA), which is important for inhibiting histone deacetylation in cancer [32]. Li et al. combined pRNA dimers with siRNA and aptamer, and they demonstrated that pRNA, which is a component of a phi29 bacteriophage, can be useful for efficient siRNA delivery. The pRNA-siRNA-aptamer was encapsulated in folate and PEG functionalized

chitosan nanoparticles. In that study, the aptamer used was FB4 for a transferrin domain, and the siRNA was c-myc [33]. Wu et al. demonstrated that functionalized cationic nanobubbles decorated with PSMA aptamers are able to carry and deliver FoxM1 siRNA to prostate cancer positive cells and xenographic tumors [34]. One recent study developed a programmable DNA nanostructure that is considered a potential system for drug delivery for non-small cell lung cancer. DNA Prism nanostructure conjugated with MUC-1 aptamers to deliver Rab26 siRNA presented a great cellular uptake efficiency and anti-tumoral activity [35].

In the field of theranostics, this approach can be useful. Theranostics is defined as nanomedicine that can deliver therapeutics to an intended region, diagnose disease, and follow the progression of disease [36]. Recently, interdisciplinary research in this field, which applies theranostics to diagnose and treat cancer, has explosively progressed. Kim et al. 2017 developed a theranostic liposomal system for carrying diagnostic Quantum Dots (QD) and therapeutic siRNA that were coupled to aptamer molecules against EGFR. The theranostic liposomes were evaluated in terms of cancer-targeted siRNA delivery and QD imaging in vitro and in vivo [11]. The anti-EGFR aptamer was used recently as a theranostic delivery system. The authors used two siRNA to provide a combinatorial anti-cancer treatment using BCL-2 siRNA, which interfere with tumor cell proliferation, and siRNA PKC-ι for inhibition of tumor cell migration. Moreover, these vehicles were able to simultaneously delivered QD provided fluorescence signals in internal organs and tumors. This study shows an efficiently delivered targeting of anti-cancer therapeutic siRNAs, as well as fluorescent QDs to tumor tissues, enabling the use of theranostic delivery systems in the treatment and fluorescence imaging of cancers [37].

In this review, we compiled several studies with different types of nanoparticles; despite different types of nanoparticles, aptamer functionalized nanoparticle loading therapeutic siRNA design can generally be done in two ways: (1) incorporated in the nanoparticle itself or (2) conjugated to the nanoparticle surface.

Over the past few years, the investment in therapeutic oligonucleotides (e.g., aptamers, siRNAs, and antisense technology) is constantly increasing. It is projected that clinical testing of aptamer-targeted drugs will materialize over the next 2–5 years However, there are several challenges in the use of aptamers as nanoparticle drivers, including the aptamer structural design, in vivo stability, immunogenicity, cellular uptake, and endosomal escape, that should be considered [16]. There is the possibility of target specificity, RNAi efficacy, and stability of siRNA-aptamers chimeras being affected by chemical modifications. Therefore, it is crucial to find an optimal alternative for each siRNA aptamers chimera to maximize its therapeutic efficacy [38].

In order to increase extracellular stability, incorporation of protective groups, such as thiol-phosphate, 20-fluoro, 20-amino, and 2 -*O*-methyl, in phosphate or sugar of nucleotides improves nuclease resistance of aptamer-siRNA chimeras [39,40]. Another possibility for improving stability is the aptamers PEG conjugation. Furthermore, it has been shown that PEG formulation reduces immune responses in vivo [41]. The thermal stability of siRNA is another parameter to be considered in gene silencing activity when it is conjugated to aptamers [38]. In this context, the computational modeling has been used to guide RNA aptamer truncations, maintaining their functionality, and preventing and minimizing therapy challenges using siRNA.

Several approaches have been developed to facilitate siRNA uptake and nanoparticle entry into endosomes. One possibility is to perform the conjugation of chimeras to short cell penetration cationic peptides (CPPs) that are important for the efficiency in intracellular transport of a variety of macromolecules [42]. Another possibility is the conjugation of the chimeras with peptides from the transactivator of transcription (TAT)region; Diao et al. demonstrated that the survivin siRNA was delivering specifically and efficiently and suppressed tumor growth in prostate cancer in vivo [43]. Inclusion of endosome-disrupting molecules, including PEI and poly (histidine), as well as fusogenic lipids and pH-sensitive lipids in aptamer-siRNA chimeras, have been useful for enhancing siRNA endosomal evasion [24,44,45]. Table 1 summarizes carriers aptamers cited in this section.


#### **Table 1.** Aptamers for delivery of siRNA.

#### **3. Cancer Immunotherapy Using Aptamers-siRNA**

Immunotherapy has emerged as the best clinical alternative for cancer treatment during the last years. It is mostly based in monoclonal antibodies targeting immunological checkpoint pathways, as well as in genetically-engineered cells that directly attack the tumor, like CAR-T cells. Despite the great success of immunotherapy, it works for 20–40% of the patients, depending on tumor type and disease progression stage [46]. So, other classes of molecules and treatments, such as those based in RNA and aptamers, appeared as good alternatives for cancer immunological treatment.

RNA-based immunotherapy is, in concept, more advantageous than those based in recombinant protein expression platforms. It has lower cost, lower immunogenicity, and better penetration in the tumor tissue, which makes it worth developing. In addition, in the last years, a number of pre-clinical and clinical data proved its efficacy, using both aptamer alone and RNAi conjugates.

Antagonistic and agonistic aptamers, acting similar to monoclonal antibodies, were developed to bind to immunomodulating molecules, such as CTLA-4, PD-1, IL-10 R, and LAG-3. The tetrameric antagonistic aptamer selected for CTLA-4, the first one to be developed, was very effective during melanoma murine model studies in vivo [47]. In the same way, multimeric structures of aptamers were shown to have a better performance for agonistic ligands, bringing them together during the activation. It is the case of OX-40 agonistic aptamer, Pratico et al. constructed a multimerized anti OX-40 aptamer using biotinylation and demonstrated a much better biologic effect than the single aptamer, with increased IFN-© production and proliferation, using peripheral blood mononuclear cells as an in vitro model of T cells' activation [48].

Some receptors of the immune system have an interesting characteristic: They get internalized after T cell activation as a feedback mechanism of controlling over-stimulation. Taking advantage of this feature, some aptamers have been developed as delivery systems to siRNA. The first one was 4-1BB aptamer used to deliver siRNA to T cells, aiming to disrupt mTOR pathway and attenuate IL-2R signaling in CD8<sup>+</sup> T cells, which, in turn, leads to CD8<sup>+</sup> T cells persistence inside the tumor site and its elimination [49]. This same aptamer was used to address siRNA for Smad-4, a protein from TGF-β signaling cascade. TGF-β is a key mediator of immune system suppression during progression. This approach was successful in suppressing the negative effect of TGF-βon infiltrating CD8<sup>+</sup> cells elicited by vaccination with tumor antigens [50].

Cluster of Differentiation (CD) aptamers also have been shown as good alternatives for RNAi delivery. CD molecules are used traditionally for identification of subpopulations of immune cells during flow cytometry studies and, as well as 4-1BB, are frequently internalized after cells activation. For example, using CD4 aptamers complexed with short hairpin RNAs (shRNA), it was possible to silence ROR©T pathway, the key for TH17 cells development, opening a possibility of treatment for a number of conditions, mainly related to autoimmune diseases [51]. In the cancer field, Soldevilla et al. used the previously developed agonistic CD40 aptamer to construct shRNA-aptamer chimeras to inhibit SMG1, a kinase that is essential for nonsense-mediated mRNA decay (NMD) initiation. Mice treated with the CD40 aptamer-shRNA chimera showed higher tumor infiltration of lymphocytes [52,53]. Table 2 summarizes aptamers-siRNAs used for immunotherapy cited in this section.


