**4. Tumor-Targeted Aptamers Complexed Directly to siRNAs and Other Agents for Cancer Therapy**

Thanks to the unique features of aptamers, such as stability and tumor penetration, its use as tools for delivery of therapeutic agents, including siRNA, has gained great attention from researchers during the last few years.

The idea of a tailored drug for the treatment of a diseased cell avoiding affecting the surrounding healthy cells is not recent, as recalled by Kruspe and Giangrande [54]. It was initially proposed by Paul Ehrlich about one century ago, when he first defined the "magic bullet" and its inherent concept of cell-surface receptor [55]. The route scientists have threaded to reach the current status has grooved a persistent tentative of diverse therapy strategies, some among them based on the conjugation of aptamer with siRNA.

Aptamers, however, due to their anionic characteristics, do not easily transit across the cell lipid bilayer, requiring adequate strategies to overcome the action of those charges, using diverse adjuvants in different structures based on lipids/liposomes, nanoparticles (as seen above), cationic polymers, and peptides.

Aptamer-siRNA conjugates, or AsiCs as they are frequently referred to, comprise an ever-increasing number of structures since 2006 when the delivery of siRNA by an aptamer was described simultaneously by two groups working with prostate membrane antigen (PSMA).

The first aptamer to be conjugated to an siRNA was to treat prostate cancer. The strategy consisted in addressing the tumor trough PSMA, a protein overexpressed in the surface of prostate cancer cells and tumor vascular endothelium, with a PSMA aptamer linked to PLK-1 and BCL-2 siRNAs. The siRNAs were responsible for disrupting the protection against apoptosis of the tumor cells. The tool was successful in eliminating a xenograft prostate cancer in a murine model [56]. After that, many other groups developed similar strategies. Interesting results were obtained for glioblastoma, the most frequent and aggressive primary brain tumor in adults, with a very poor prognosis. Expression and activation of the signal transducer and activator of transcription-3 (STAT3) has been reported as a key regulator of this kind of tumor, so a chimera aptamer antagonistic for PDGFRbr (a receptor overexpressed in glioblastoma, as well as other tumor types) carrying siRNA for STAT 3 was designed. This chimera was able to reduce the anti-apoptotic factors Poly (ADP-ribose) polymerase PARP and BCL-xL, inducing cell death in two glioblastoma lineages in vitro. Moreover, Gint4.T-STAT3 chimera treatment induced significant reduction of tumor growth rate in comparison to control group, as well reduced pro-tumoral factors in a xenograft mouse model of glioblastoma [57]. Similar results were obtained for HER+ breast cancer using a HER-2 sense and antisense bivalent aptamer conjugated to EGFR siRNA. It was able to be delivered to breast cancer cells overexpressing HER-2 and the

EGFR gene was efficiently downregulated in vitro, whilst a xenografted tumor growth was supressed in vivo [58]. Another study from the same group showed the performance of a bivalent HER-2-HER-3 aptamer with an EGFR siRNA between them. It was even better in reducing the expression of all three receptors, in inducing cell cycle arrest and apoptosis in vitro and inhibiting tumor growth with a little of target effect in vivo [59]. Aptamer-siRNA chimeras were also used to overcome multidrug resistance by Jeong et al. They engineered a mucin multiaptamer (E18 units) conjugated to RNAsi for BCL-2, with the intercalation of doxorubicin (DOX) between the double helix of the aptamers. Promising results were obtained using this construct to treat MUC-1 overexpressing MCF-7 breast cancer cells. The complex was efficient in delivering doxorubicin to multi drug resistant MCF-7 cells because of increased endocytosis efficiency due to the cluster effect. BCL-2 RNAsi acted synergistically with DOX, increasing the sensitivity of cells for apoptosis and, in turn, decreasing cell viability [60].

An interesting work has used DOX to inactivate cancer stem cells (CSC) by conjugating this drug to engineered RNA aptamers raised against CSC surface marker epithelial cellular adhesion molecule (EpCAM). A modified DNA-RNA hybrid EpCAM aptamer (10-bp GC at stem region) was loaded with 2,5 molecules of DOX per aptamer and this conjugate was capable to release approximated 89.2% of the intercalated DOX just after endocytosis. The authors proved the release of DOX was dependent on low pH that in turn limited the availability of the drug in systemic circulation and tissue interstitium (physiological pH) to affect sensitive organs. Additionally, the complex Apt-DOX was able to target and deliver DOX to EpCAM positive HT29 colorectal cancer cells in dose and time dependent manner and lead to the accumulation and retention of the drug in the cell nucleus. All these characteristics made this construct very efficient in controlling tumor growth overcoming chemoresistance in both tumorsphere formation assay and immunodeficient mice with LDA xenotransplanted with CSC. The aptamer deliver approach developed by Xiang et al. could in fact improve therapeutic index delivering a sufficient drug dose to the critical subcellular target for enough time to eliminate precisely CSCs [61]. The same group extended the investigation to new approaches to tackle cancer stem cells using EpCAM aptamer. They use the aptamer as a driver to survivin RNAi in colorectal cancer cells, both in vitro as well as in colorectal tumor bearing mice. The combination of the surivin downregulation with the action of 5-fluorouacil cytotoxic chemotherapeutic could increase the lethality of colorectal cancer stem cells, as well as tumor control, in the colorectal cancer xenograph model and, consequently, improvement of animal survival [62].

A proof of concept of an approach using an aptamer-liposomal-DOX to target HER3ECD (epitope to trastuzumab binding epitope antigen) with minimum undesirable toxicity is described by Dou Xi et al. The strategy was to conjugate a single-stranded DNA aptamer against HER3 to liposome to improve targeted delivery with less unspecific action of DOX. The aptamer raised against HER3CD was able to bind to HER3 in MCF-7 HER3<sup>+</sup>, BT 474HER3<sup>+</sup> but not in 293T HER3<sup>−</sup> cells. This aptamer originates the apt-Lip-DOX conjugate that in fact increased the sustained drug release and was able to increase the growth inhibition of MCF-7 HER3<sup>+</sup> and BT 474HER3<sup>+</sup> cells when compared to liposome-DOX and DOX alone challenge. The Apt-Lip-DOX formulation had many advantages when compared to Lip-DOX treatment. There was a higher uptake and retention by MCF-7 tumors in mice. The survival rate of the animals group treated with the apt-Lip-DOX 60 days after chemotherapy was 40% against 0% of the DOX group attained by 36th day after treatment. It is interesting to notice there was any lethality in the Apt-Lip-DOX treated control group that means a very low toxicity of this construct. Additionally, liver and cardio toxicity in the Apt-Lip-DOX treated group was compared to the control NaCl treated group. Finally, the author have demonstrated that the use of aptamers as a driver to target cells unequivocally reduced the availability of DOX in tissues (reduced biodistribution) other than tumor, reducing significantly the cardio toxicity after chemotherapy when compared to the formulation without aptamer [63].

Prusty et al. (2018) developed a very elegant photo-switchable hybrid-aptameric nanoconstruct that efficiently release DOX at high concentration to HGFR (hepatocyte growth factor receptor–cMet) expressing cells. The HyApNc-DOX nanoconstruct was obtained by the combination of two lipid

self-assembly single motifs. The first motif was constituted by a lipid-functionalized aptamer trCLN3 with four lipid-modified dU-phosphoramidite 1 at the 5 end of the aptamer. The so-called trCLN3-L4 retained nanomolar affinity to the cMet receptor in tumor cells despite the modification. The second motif was a lipid functionalized 5 -CG rich hairpin ODN designed to carry DOX by intercalation but with a 2 ,6 -dimethylzobenzene (DMAB) photoswitch incorporated in the hairpin nanoscaffold. After UV irradiation, DMAB is responsible for the controlled release of DOX inside cell. A lipid-mediated self-assembly approach to build a multi component supramolecular structure resulted in an improved survival to serum nucleases attack and in a better efficiency in the uptake by the target cell and also a precise subcellular distribution of DOX to the cell nucleus, after endocytosis, when compared to the native aptamer [64].

Another approach using aptamers to deliver DOX was developed to target Glioblastoma multiforme tumor cells. A GMT-3 ssDNA aptamer raised against glioblastoma multiforme A-172 cells demonstrates a high affinity to different glioblastoma cell lines (Kd, ~75 nM). Aptamer GMT-3 charged with DOX (molar ratio 1.2 DOX per aptamer) could selectively bind to and inactivate A-172 glioblastoma cells in comparison to the effect noticed in MCF-7 control cells [65].

A great challenge in the treatment of Glioblastoma using chemotherapeutics is to overcome the blood brain barrier (BBB) and to avoid chemotherapeutic drug deliver to the non-tumor tissues. Luo et al. (2017) developed a nanoconjugate delivery system using AS1411 aptamer as a driver to target nucleolin receptor that is overexpressed in glioblastoma U87 MG cells and neo-vascular endothelial cells in way to deliver chemotherapeutics with more precision. AS 1411-Nucleolin complex undergoes endocytosis inhibiting DNA synthesis inducing apoptosis and also inhibiting angiogenesis, that means it has dual targets. The authors incorporated a more efficient cytotoxic drug to AS1411 synthetizing a poly (l-*c*-glutamyl-glutamine)-paclitaxel nanoconjugate (PGGPTX) to achieve an improvement of aqueous solubility and permanence in plasma. All the results obtained by the group demonstrated that the presence of AS1441 aptamer in the complex AS1411-PGG-PTX improved significantly the U87 MG, as well as neo-vascular endothelial cells uptake and internalization, U87 MG spheroid tumor penetration, and U87 MG cell growth inhibition. The same effect could be demonstrated in vivo with an enhanced accumulation of AS1411-PGG-PTX nanoconjugates in glioblastoma cells, its extended retention time in circulation, and its pronounced penetration in glioblastoma tumors. The authors observed that AS1411-PGG-PTX was able to promote an increase in the survival of the tumor bearing mice and an efficient PCX delivery to glioblastoma tissue with a consequently strong cytotoxic effect [66].

One interesting strategy was used by Balasubramanyam and collaborators in a recently published work, where they designed aptamer-siRNA chimeras with great specificity to cancer expressing EpCAM that can deliver siRNAs against chosen oncogenes. In the case, PLK1, BCL2, and STAT3, three important proteins with high relevance in tumor growth, were selected. They tested various chimeras with positive results in cancer cell lines of breast, lung, head, neck, liver, and retinoblastoma [67].

Li et al. (2017) demonstrated an interesting feature of the aptamers paving the way to the action of neoadjuvant cytotoxic drugs, not necessarily carrying drugs molecules in their own structure. They selected a new ssDNA named HL-1 with high affinity and specificity to Maver-I lymphoma cells (Kd = 70 pmol/L). The aptamer presents a G-quadruplex structure and, once internalized by endocytosis, promoted cell cycle arrest in S-phase in around half of cell population after three days of lymphoma cells treatment. They exploit this effect in a combined treatment of lymphoma cells with a cytotoxic cytarabine. The lethal action of cytarabine is predominant over lymphoma cells in S-phase, and they show that several rounds of aptamer treatment was able to synergistically sensitize these cells to the lethal effect of cytarabine [68].

Pancreatic ductal adenocarcinoma (PDAC) could receive a great benefit from an aptamer target drug deliver approach because it is one with the worst prognosis among all other cancers. The limitation of therapeutic drug treatment dose imposed by the side effects resulted from the non-specificity of the available cancer chemotherapeutics contributes significantly to a poor prognosis. Levy and Kratchmer (2018) proposed a new treatment approach using the Waz aptamer (anti-transferrin receptor–TfR) and E07 aptamer (anti-epidermal growth factor—EGFR), two receptors overexpressed in PDAC that are internalized by clathrin mediated endocytosis. The product of the conjugation of these aptamers with thiol-reactive membrane-permeable MMAE (MC-VC-PAB-MMAE) bearing a valine-citrulline linker or the membrane-impermeable auristatin derivative MMAF (MC-MMAF) was tested in Panc-1, MIA PaCa-2, and BxPC3 PDAC cell lines, with promising results [69].

Finally, the MUC1 aptamers were also used to deliver chitosan nanoparticles containing both docetaxel and siRNAs for a co-delivery/co-treatment approached. The conjugation of the aptamer to the nanoparticle increased specificity and cellular uptake of the nanoconjugate, whereas a significant impact of the combination therapy was confirmed in terms of cell viability. Furthermore, successful silencing was confirmed. Although this was a preliminary study conducted in vitro, it also demonstrates the potential of this type of approach [70]. Table 3 summarizes all works cited in this section.


**Table 3.** Aptamers for delivery SiRNA/chemotherapeutics for cancer therapy.


#### **Table 3.** *Cont.*

### **5. Concluding Remarks**

Today, there are 42 studies in clinical trials using the term "aptamer", and 64 completed or ongoing studies using the term "siRNA" for treating medical conditions, including: cancer, HIV, and diseases of the immune system (www.clinicaltrial.gov), which indicates that these two therapeutic agents are still on the frontier of knowledge and are tendency in the global market. In a study of market analysis, siRNA and aptamers are the new drugs that biopharmaceutical industries are investing, and the cancer is the most studied disease using this strategy. The global aptamer market was valued at nearly \$1.0 billion USD in 2016 and is expected to expand with a CAGR of 20.0% over the period from 2017 to 2025 to attain the value of \$5.0 billion USD by the end of 2025. This growth is attributable to technology advances and the introduction of companies at commercial panel. The main players operating in the global aptamer market are AM Biotech (Houston, TX, USA), Aptamer Group (York, UK), Aptamer Sciences Inc. (Gyeongsangbuk-do, Korea), Aptagen LLC (Jacobus, PA, USA), and Base Pair Biotechnologies (Pearland, TX, USA). In regard to siRNA, the therapeutics market is expected to expand significantly until 2025, since the first product approved by FDA is currently available in the market. Major players operating in the global small interfering RNA (siRNA) therapeutics market are GE Dharmacon Lafayette, USA), OPKO Health, Inc. (Miami, FL, USA), Alnylam Pharmaceuticals (Cambridge, MA, USA), Arrowhead Research Corp (Pasadena, CA, USA), Sanofi Genzyme (Cambridge, MA, USA), Genecon Biotechnologies Co., Ltd. (Baesweiler, Germany), Arbutus Biopharma Corp (Burnaby, Canada), Silence Therapeutics AG (London, UK), and Sylentis S.A. (Madrid, Spain) (www.grandviewresearch.com).

Ever since our last revision of the field in 2017 [71], more than 50 new works have been published and revised in this manuscript, which show the potential of this approach. Furthermore, a number of new works have appeared related to nanoparticle formulations of siRNA and other RNA forms, using different types of nanoparticles, and carried successfully by aptamers, whilst some approaches have favored the use of siRNAs in conjunction with chemotherapy agents, with very promising results. A review of some of these technologies in a graphic form is presented in the figure below (Figure 1).

**Figure 1.** Uses of aptamers in therapeutic delivery.

Harnessing the targeting potential of aptamers, together with the silencing power of siRNAs and a long proved cytotoxic effect of chemotherapeutic agents, and bringing them together into a single therapeutic modality, has shown a great co-operative effect. It offers a therapy delivered directly and specifically to the tumor cell, with important proteins for tumor progression or survival silenced, whilst DNA acting cytotoxic agents act on tumor DNA, causing a specific therapy that has the potential to outmatch other approaches that focus on a single modalities.

**Author Contributions:** Conceptualization, S.M. and C.E.B.d.A.; writing—original draft preparation: Aptamer in the delivery of siRNA: A.P.D.A.B., Immunotherapy Using Aptamers-siRNA: P.C.d.C.N. and D.S., Aptamers-siRNA Chimeras: C.E.B.d.A., P.C.d.C.N. and D.S.; writing—review and editing, S.M.; supervision, S.M.

**Funding:** This work was supported by Instituto de Tecnologia em Imunobiológicos (Bio-Manguinhos).

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