A responsive disulfide bond switch aptamer prodrug exhibiting enhanced stability and anticancer efficacy.

Aptamers have shown significant potential in treating diverse diseases. However, challenges such as stability and drug delivery limited their clinical application. In this paper, the development of AS1411 prodrug-type aptamers for tumor treatment was introduced. A Short oligonucleotide was introduced at the end of the AS1411 sequence with a disulfide bond as responsive switch. The results indicated that the aptamer prodrugs not only enhanced the stability of the aptamer against nuclease activity but also facilitated binding to serum albumin. Furthermore, in the reducing microenvironment of tumor cells, disulfide bonds triggered drug release, resulting in superior therapeutic effects in vitro and in vivo compared to original drugs. This paper proposes a novel approach for optimizing the structure of nucleic acid drugs, that promises to protect other oligonucleotides or secondary structures, thus opening up new possibilities for nucleic acid drug design.

Innovative gelatin-based micelles with AS1411 aptamer targeting and reduction responsiveness for doxorubicin delivery in tumor therapy.

Herein, we constructed innovative reduction-sensitive and targeted gelatin-based micelles for doxorubicin (DOX) delivery in tumor therapy. AS1411 aptamer-modified gelatin-ss-tocopherol succinate (AGSST) and the control GSST without AS1411 modification were synthesized and characterized. Antitumor drug DOX-containing AGSST (AGSST-D) and GSST-D nanoparticles were prepared, and their shapes were almost spherical. Reduction-responsive characteristics of DOX release in vitro were revealed in AGSST-D and GSST-D. Compared with non-targeted GSST-D, AGSST-D demonstrated better intracellular uptake and stronger cytotoxicity against nucleolin-overexpressed A549 cells. Importantly, AGSST-D micelles showed more effective killing activity in A549-bearing mice than GSST-D and DOX⋅HCl. It was revealed that AGSST-D micelles had no obvious systemic toxicity. Overall, AGSST micelles would have the potential to be an effective drug carrier for targeted tumor therapy.

An anti-GD2 aptamer-based bifunctional spherical nucleic acid nanoplatform for synergistic therapy targeting MDM2 for retinoblastoma.

Retinoblastoma (RB) is a type of pediatric solid tumor in the fundus. The lack of precision therapies combined with the difficulty of delivering small interfering RNA (siRNA) into the eyes means that there is currently no nucleic acid-based therapy for RB in clinical practice. Here, we reported on anti-GD2 and glutathione-responsive spherical nucleic acids (SNAs), loaded with siRNA and the inhibitor NVP-CGM097, which jointly blocked the oncogenic factor n in RB cells (Y79 and WERI-RB-1). The SNAs were formed through the self-assembly of bifunctional cholesterol amphiphiles containing aptamers that specifically targeted GD2-positive RB cells, allowing for the formation of an SNA with a dense DNA shell. The aptamer/siRNA component functioned both as a carrier and a payload, enhancing the specific recognition and delivery of both components and constituting an active agent for MDM2 regulation. Following SNA endocytosis by RB cells, siRNA and NVP-CGM097 were released from the SNA particles by glutathione, which synergistically blocked the MDM2-p53 pathway, increasing p53 protein content and inducing cell apoptosis. This study showed a potent antitumor effect following intravitreal injection of SNAs in Y79 tumor-bearing mice through clinical manifestation and tumor pathological analysis. In hematological analysis and hepatotoxicity assays, SNAs were safer for mice than melphalan, the favored drug for treating RB in clinical practice. Our results illustrated the potential of intravitreally injected SNAs as a precision medicine for treating RB.

Construction of aptamer-siRNA chimera and glutamine modified carboxymethyl-ß-cyclodextrin nanoparticles for the combination therapy against lung squamous cell carcinoma.

Combination therapy has become the most important treatment for advanced non-small cell lung cancer (NSCLC), which can significantly improve the prognosis of patients. However, poor targeting and adverse reactions limited its clinical application. Here, we constructed an AS1411 aptamer-programmed cell death ligand-1 (PD-L1) siRNA chimera/polyethylenimine/glutamine/ß-cyclodextrin/doxorubicin (Chimera/ PEI/Gln/ß-CD/DOX) nanoparticle for the combination therapy (chemotherapy combined with immunotherapy). Scanning electron microscopy showed that PEI/Gln/ß-CD/DOX nanoparticle was conical, with a diameter of about 250-500 nm. AS1411 aptamer-PD-L1 siRNA chimera can effectively bind NSCLC cells and inhibit PD-L1 expression, further activating T cells and CD8+T cells. Glutamine modification effectively promoted the doxorubicin uptake by cancer cells and induced their apoptosis. Animal experiments showed that our nanoparticles effectively treated the transplanted tumor, and the adverse reactions were reduced. Compared with the Aptamer/ß-CD/DOX group, the volume and ki-67 index of transplanted tumors in the Chimera/ß-CD/DOX group were significantly decreased, while the apoptosis ratio was increased. Immunohistochemical results showed that Compared with the Aptamer/ß-CD/DOX group, the number of T cells and CD8+T cells in the Chimera/ß-CD/DOX group was increased by 1.34 and 1.41 times. Glutamine modification enhanced the chemotherapeutic efficacy and anti-tumor immune response in vivo. Our study provided a new method for the combination therapy of lung squamous cell carcinoma.

Directing in Vitro Selection towards G-quadruplex-forming Aptamers to Inhibit HMGB1 Pathological Activity.

In the search for novel, effective inhibitors of High-Mobility Group Box1 (HMGB1)-a protein involved in various inflammatory and autoimmune diseases as well as in cancer-we herein discovered a set of anti-HMGB1 G-quadruplex(G4)-forming aptamers by using an in vitro selection procedure applied to a doped library of guanine-rich oligonucleotides. The selected DNA sequences were then studied in a pseudo-physiological buffer mimicking the extracellular medium, where HMGB1 exerts its pathological activity, using spectroscopic, electrophoretic, and chromatographic techniques. All the oligonucleotides proved to fold into monomeric G4s and in some cases also dimeric species, stable at physiological temperature. Remarkably, the protein preferentially recognized the sequences forming dimeric parallel G4 structures, as evidenced by a properly designed chemiluminescent binding assay which also highlighted a good selectivity of these aptamers for HMGB1. Moreover, all aptamers showed anti-HMGB1 activity, inhibiting protein-induced cell migration. The acquired data allowed identifying L12 as the best anti-HMGB1 aptamer, featured by high thermal and enzymatic stability, no toxicity at least up to 5 µM concentration on healthy cells, along with potent anti-HMGB1 activity (IC50 ca. 28 nM) and good binding affinity for the protein, thus indicating it as a very promising lead candidate for in vivo studies.

[Covalently conjugated DNA aptamer with doxorubicin as in vitro model for effective targeted drug delivery to human glioblastoma tumor cells]. / Kovalentno kon"yugirovannyi DNK-aptamer s doksorubitsinom kak in vitro model' effektivnogo adresnogo vozdeistviya na opukholevye kletki glioblastomy cheloveka.

Targeted delivery of chemotherapeutic agents with aptamers is a very effective method increasing therapeutic index compared to non-targeted drugs. OBJECTIVE: To study the effectiveness of in vitro therapeutic effect of covalently conjugated GR20 DNA aptamer with doxorubicin on glioblastoma cells compared to reference culture of human fibroblasts. MATERIAL AND METHODS: A Sus/fP2 cell culture was obtained from glioblastoma tissue sample to analyze the effectiveness of conjugate. A linear culture of human dermal fibroblasts (mesenchymal stem cells) DF1 was used as a control. To assess antiproliferative activity of covalently conjugated GR20 aptamer with doxorubicin, we used the MTS test. The Cell Index was measured using the xCelligence S16 cell analyzer assessing viability of cell cultures by recording changes in real time. RESULTS: Human glioblastoma Sus/fP2 cells reduce own proliferative potential by 80% when exposed to doxorubicin (0.5 µM, 72 hours, MTS test), by 9% when exposed to GR20 aptamer (10 µM, 72 hours, MTS test) and by 26% when exposed to covalently conjugated DOX-GR20 (0.5 µM, 72 hours, MTS test). A long-term study of proliferative potential of Sus/fP2 cells on the xCelligence S16 analyzer revealed a significant decrease in the number of cells under the effect of doxorubicin and covalently conjugated DOX-GR20. Effectiveness of covalently conjugated DOX-GR20 is halved. GR20 aptamer at a concentration of 10 µM and its conjugate with doxorubicin DOX-GR20 at a concentration of 1 µM have no negative effect on cells of the control culture of DF1 fibroblasts, while doxorubicin is toxic for these cells. MTS test and xCelligence S16 cell analyzer found no decrease in metabolic activity of DF1 cells and their ability to proliferate. CONCLUSION: We established obvious antiproliferative effect of covalent conjugate DOX-GR20 on continuous human glioblastoma cell culture Sus/fP2 without toxic effect on the reference culture (dermal fibroblasts DF1).

Targeting vimentin: a multifaceted approach to combatting cancer metastasis and drug resistance.

This comprehensive review explores vimentin as a pivotal therapeutic target in cancer treatment, with a primary focus on mitigating metastasis and overcoming drug resistance. Vimentin, a key player in cancer progression, is intricately involved in processes such as epithelial-to-mesenchymal transition (EMT) and resistance mechanisms to standard cancer therapies. The review delves into diverse vimentin inhibition strategies. Precision tools, including antibodies and nanobodies, selectively neutralize vimentin's pro-tumorigenic effects. DNA and RNA aptamers disrupt vimentin-associated signaling pathways through their adaptable binding properties. Innovative approaches, such as vimentin-targeted vaccines and microRNAs (miRNAs), harness the immune system and post-transcriptional regulation to combat vimentin-expressing cancer cells. By dissecting vimentin inhibition strategies across these categories, this review provides a comprehensive overview of anti-vimentin therapeutics in cancer treatment. It underscores the growing recognition of vimentin as a pivotal therapeutic target in cancer and presents a diverse array of inhibitors, including antibodies, nanobodies, DNA and RNA aptamers, vaccines, and miRNAs. These multifaceted approaches hold substantial promise for tackling metastasis and overcoming drug resistance, collectively presenting new avenues for enhanced cancer therapy.

Aptamer-Modified Tetrahedral DNA Nanostructures as Drug Delivery System for Cancer Targeted Therapy.

Improving the selective delivery and uptake efficiency of chemotherapeutic drugs remains a challenge for cancer-targeted therapy. In this work, a DNA tetrahedron is constructed as a targeted drug delivery system for efficient delivery of doxorubicin (Dox) into cancer cells. The DNA tetrahedron is composed of a tetrahedral DNA nanostructure (TDN) with two strands of AS1411 aptamer as recognition elements which can target the nucleolin protein on the cell membrane of cancer cells. The prepared DNA tetrahedron has a high drug-loading capacity and demonstrates pH-responsive Dox release properties. This enables efficient delivery of Dox into targeted cancer cells while reducing side effects on nontarget cells. The proposed drug delivery system exhibits significant therapeutic efficacy in vitro compared to free Dox. Accordingly, this work provides a good paradigm for developing a targeted drug delivery system for cancer therapy based on DNA tetrahedrons.

Tools and techniques for the discovery of therapeutic aptamers: recent advances.

INTRODUCTION: The pursuit of novel therapeutic agents for serious diseases such as cancer has been a global endeavor. Aptamers characteristic of high affinity, programmability, low immunogenicity, and rapid permeability hold great promise for the treatment of diseases. Yet obtaining the approval for therapeutic aptamers remains challenging. Consequently, researchers are increasingly devoted to exploring innovative strategies and technologies to advance the development of these therapeutic aptamers. AREAS COVERED: The authors provide a comprehensive summary of the recent progress of the SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique, and how the integration of modern tools has facilitated the identification of therapeutic aptamers. Additionally, the engineering of aptamers to enhance their functional attributes, such as inhibiting and targeting, is discussed, demonstrating the potential to broaden their scope of utility. EXPERT OPINION: The grand potential of aptamers and the insufficient development of relevant drugs have spurred countless efforts for stimulating their discovery and application in the therapeutic field. While SELEX techniques have undergone significant developments with the aid of advanced analysis instruments and ingeniously updated aptameric engineering strategies, several challenges still impede their clinical translation. A key challenge lies in the insufficient understanding of binding conformation and susceptibility to degradation under physiological conditions. Despite the hurdles, our opinion is optimistic. With continued progress in overcoming these obstacles, the widespread utilization of aptamers for clinical therapy is envisioned to become a reality soon.

Insights on the molecular mechanisms of cytotoxicity induced by AS1411 linked to folate-functionalized DNA nanocages in cancer cells.

Self-assembled multivalent DNA nanocages are an emerging class of molecules useful for biomedicine applications. Here, we investigated the molecular mechanisms of cytotoxicity induced by AS1411 free aptamer, AS1411-linked nanocages (Apt-NCs) and nanocages harboring both folate and AS1411 functionalization (Fol-Apt-NCs) in HeLa and MDA-MB-231 cancer cell lines. The three treatments showed different cytotoxic efficacy and Fol-Apt-NCs resulted the most effective in inhibiting cell proliferation and inducing apoptotic pathways and ROS activation in both HeLa and MDA-MB-231 cells. RNA-seq analysis allowed to identify biological functions and genes altered by the various treatments, depending on the AS1411 route of intracellular entry, highlighting the different behavior of the two cancer cell lines. Notably, Fol-Apt-NCs altered the expression of a subset of genes associated to cancer chemoresistance in MDA-MB-231, but not in HeLa cells, and this may explain the increased chemosensitivity to drugs delivered through DNA nanocages of the triple-negative breast cancer cells.

Screening a DNA Aptamer Specifically Targeting Integrin ß3 and Partially Inhibiting Tumor Cell Migration.

Due to its key roles in malignant tumor progression and reprograming of the tumor microenvironment, integrin ß3 has attracted great attention as a new target for tumor therapy. However, the structure-function relationship of integrins ß3 remains incompletely understood, leading to the shortage of specific and effective targeting probes. This work uses a purified extracellular domain of integrin ß3 and integrin ß3-positive cells to screen aptamers, specifically targeting integrin ß3 in the native conformation on live cells through the SELEX approach. Following meticulous truncation and characterization of the initial aptamer candidates, the optimized aptamer S10yh2 was produced, exhibiting a low equilibrium dissociation constant (Kd) in the nanomolar range. S10yh2 displays specific recognition of cancer cells with varying levels of integrin ß3 expression and demonstrates favorable stability in serum. Subsequent analysis of docking sites revealed that S10yh2 binds to the seven amino acid residues located in the core region of integrin ß3. The S10yh2 aptamer can downregulate the level of integrin heterodimer αvß3 on integrin ß3 overexpressed cancer cells and partially inhibit cell migration behavior. In summary, S10yh2 is a promising probe with a small size, simple synthesis, good stability, high binding affinity, and selectivity. It therefore holds great potential for investigating the structure-function relationship of integrins.

Discovery of Aptamers and the Acceleration of the Development of Targeting Research in Ophthalmology.

Aptamers are widely applied to diagnosis and therapy because of their targeting. However, the current progress of research into aptamers for the treatment of eye disorders has not been well-documented. The current literature on aptamers was reviewed in this study. Aptamer-related drugs and biochemical sensors have been evaluated for several eye disorders within the past decade; S58 targeting TGF-ß receptor II and pegaptanib targeting vascular endothelial growth factor (VEGF) are used to prevent fibrosis after glaucoma filtration surgery. Anti-brain-derived neurotrophic factor aptamer has been used to diagnose glaucoma. The first approved aptamer drug (pegaptanib) has been used to inhibit angiogenesis in age-related macular degeneration (AMD) and diabetic retinopathy (DR), and its efficacy and safety have been demonstrated in clinical trials. Aptamers, including E10030, RBM-007, AS1411, and avacincaptad pegol, targeting other angiogenesis-related biomarkers have also been discovered and subjected to clinical trials. Aptamers, such as C promoter binding factor 1, CD44, and advanced end products in AMD and DR, targeting other signal pathway proteins have also been discovered for therapy, and biochemical sensors for early diagnosis have been developed based on aptamers targeting VEGF, connective tissue growth factor, and lipocalin 1. Aptamers used for early detection and treatment of ocular tumors were derived from other disease biomarkers, such as CD71, nucleolin, and high mobility group A. In this review, the development and application of aptamers in eye disorders in recent years are systematically discussed, which may inspire a new link between aptamers and eye disorders. The aptamer development trajectory also facilitates the discovery of the pathogenesis and therapeutic strategies for various eye disorders.

Aptamers Targeting the PD-1/PD-L1 Axis: A Perspective.

Aptamers have emerged in recent years as alternatives to antibodies or small molecules to interfere with the immune check points by blocking the PD-1/PD-L1 interactions and represent an interesting perspective for immuno-oncology. Aptamers are RNA or DNA nucleotides able to bind to a target with high affinity, with the target ranging from small molecules to proteins and up to cells. Aptamers are identified by the SELEX method that can be modified for specific purposes. The range of applications of aptamers covers therapy as well as new alternative assay technologies similar to ELISA. Aptamers' limited plasma stability can be managed using delivery strategies. The goal of this Perspective is to give an overview of the current development of aptamers targeting the most studied immune checkpoint modulators, PD-1 and PD-L1, and analogous strategies with aptamers for other immuno-related targets.

Highly-specific aptamer targeting SARS-CoV-2 S1 protein screened on an automatic integrated microfluidic system for COVID-19 diagnosis.

Variants of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) have evolved such that it may be challenging for diagnosis and clinical treatment of the pandemic coronavirus disease-19 (COVID-19). Compared with developed SARS-CoV-2 diagnostic tools recently, aptamers may exhibit some advantages, including high specificity/affinity, longer shelf life (vs. antibodies), and could be easily prepared. Herein an integrated microfluidic system was developed to automatically carry out one novel screening process based on the systematic evolution of ligands by exponential enrichment (SELEX) for screening aptamers specific with SARS-CoV-2. The new screening process started with five rounds of positive selection (with the S1 protein of SARS-CoV-2). In addition, including non-target viruses (influenza A and B), human respiratory tract-related cancer cells (adenocarcinoma human alveolar basal epithelial cells and dysplastic oral keratinocytes), and upper respiratory tract-related infectious bacteria (including methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae), and human saliva were involved to increase the specificity of the screened aptamer during the negative selection. Totally, all 10 rounds could be completed within 20 h. The dissociation constant of the selected aptamer was determined to be 63.0 nM with S1 protein. Limits of detection for Wuhan and Omicron clinical strains were found to be satisfactory for clinical applications (i.e. 4.80 × 101 and 1.95 × 102 copies/mL, respectively). Moreover, the developed aptamer was verified to be capable of capturing inactivated SARS-CoV-2 viruses, eight SARS-CoV-2 pseudo-viruses, and clinical isolates of SARS-CoV-2 viruses. For high-variable emerging viruses, this developed integrated microfluidic system can be used to rapidly select highly-specific aptamers based on the novel SELEX methods to deal with infectious diseases in the future.

DNA Aptamer Beacon Probe (ABP) for Monitoring of Adenosine Triphosphate Level in SW480 Cancer Cells Treated with Glycolysis Inhibitor 2-Deoxyglucose.

Early cancer screening enables timely detection of carcinogenesis, and aids in prompt clinical intervention. Herein, we report on the development of a simple, sensitive, and rapid fluorometric assay based on the aptamer probe (aptamer beacon probe, ABP) for monitoring the energy-demand biomarker adenosine triphosphate (ATP), an essential energy source that is released into the tumor microenvironment. Its level plays a significant role in risk assessment of malignancies. The operation of the ABP for ATP was examined using solutions of ATP and other nucleotides (UTP, GTP, CTP), followed by monitoring of ATP production in SW480 cancer cells. Then, the effect of a glycolysis inhibitor, 2-deoxyglucose (2-DG), on SW480 cells was investigated. The stability of predominant ABP conformations in the temperature range of 23-91 °C and the effects of temperature on ABP interactions with ATP, UTP, GTP, and CTP were evaluated based on quenching efficiencies (QE) and Stern-Volmer constants (KSV). The optimized temperature for best selectivity of ABP toward ATP was 40 °C (KSV = 1093 M-1, QE = 42%). We have found that the inhibition of glycolysis in SW480 cancer cells by 2-deoxyglucose resulted in lowering of ATP production by 31.7%. Therefore, monitoring and modulation of ATP concentration may aid in future cancer treatment.

Identification and characterization of aptameric inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation, and tissue remodeling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used the systematic evolution of ligands by exponential enrichment to develop ssDNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models.

In Situ Activation of the Receptor Aggregation for Cell Apoptosis by an AI-Au Intelligent Nanomachine via Tumor Extracellular Acidity.

Polyvalent ligand-induced cell receptor aggregation is closely related to cell behavior regulation. At present, most of the means to induce receptor aggregation rely on external stimuli such as light, heat, and magnetic fields, which may cause side effects to normal cells. How to achieve receptor aggregation on the cancer cell surface to achieve cell apoptosis selectively is still a challenge. Therefore, by taking advantage of the unique property of cancer cells' slightly acidic microenvironment, an easy-to-use apoptosis-inducing mode for the in situ activation of cell surface nucleolin clustering has been developed, which not only opened a new channel for nucleolin receptor aggregation to regulate cell function and further development but also avoided damage to normal cells, providing a new strategy for tumor treatment. Dual functional ssDNA (AS1411 aptamer and pH-responsive I-strand sequence) was modified on the surface of gold nanoparticles (AuNPs) to fabricate AI-Au intelligent nanomachines. Then, the specific binding on cancer cells and aggregation of the nucleolin receptors can be achieved via the formation of an i-Motif structure among adjacent AuNPs under the acidic microenvironment. The result showed that AI-Au nanomachines mediated nucleolin cross-linking on the cell surface, resulting in a cytotoxic effect of approximately 60%. Experiments such as calcein-AM/PI staining, nuclear dye staining, and flow cytometry demonstrated that cell apoptosis became more evident with the increase of acidity under the cell surface microenvironment. Immunofluorescence imaging further confirmed the Cyt-c/caspase-3 apoptosis pathway induced by AI-Au nanomachines. The proposed strategy used for specific cancer cell apoptosis by the in situ activation of tumor cell membrane receptor aggregation is inexpensive and simple to use, which not only provides a new means of nucleolin receptor aggregation for regulating cell function but also offers a new strategy for tumor treatment with reduced side effect to normal cells. This work is significant for comprehending the ligand-induced receptor aggregation process and can lead to the development of a promising anticancer drug.

Multi-Catcher Polymers Regulate the Nucleolin Cluster on the Cell Surface for Cancer Therapy.

Cell signal transduction mediated by cell surface ligand-receptor is crucial for regulating cell behavior. The oligomerization or hetero-aggregation of the membrane receptor driven by the ligand realizes the rearrangement of apoptotic signals, providing a new ideal tool for tumor therapy. However, the construction of a stable model of cytomembrane receptor aggregation and the development of a universal anti-tumor therapy model on the cellular surface remain challenging. This work describes the construction of a "multi-catcher" flexible structure GC-chol-apt-cDNA with a suitable integration of the oligonucleotide aptamer (apt) and cholesterol (chol) on a polymer skeleton glycol chitosan (GC), for the regulation of the nucleolin cluster through strong polyvalent binding and hydrophobic membrane anchoring on the cell surface. This oligonucleotide aptamer shows nearly 100-fold higher affinity than that of the monovalent aptamer and achieves stable anchoring to the plasma membrane for up to 6 h. Moreover, it exerts a high tumor inhibition both in vitro and in vivo by activating endogenous mitochondrial apoptosis pathway through the cluster of nucleolins on the cell membrane. This multi-catcher nano-platform combines the spatial location regulation of cytomembrane receptors with the intracellular apoptotic signaling cascade and represents a promising strategy for antitumor therapy.

Bifunctional G-Quadruplex Aptamer Targeting Nucleolin and Topoisomerase 1: Antiproliferative Activity and Synergistic Effect of Conjugated Drugs.

As a counterpart to antibody-drug conjugates (ADCs), aptamer-drug conjugates (ApDCs) have been considered a promising strategy for targeted therapy due to the various benefits of aptamers. However, an aptamer merely serves as a targeting ligand in ApDCs, whereas the antibody enables the unexpected therapeutic efficacy of ADCs through antibody-dependent cellular cytotoxicity (ADCC). In this study, we developed a tumor-specific aptamer with an effector function and used it to confirm the feasibility of more potent ApDCs. First, we designed a nucleolin (NCL)-binding G-quadruplex (GQ) library based on the ability of NCL to bind to telomeric sequences. We then identified a bifunctional GQ aptamer (BGA) inhibiting the catalytic activity of topoisomerase 1 (TOP1) by forming an irreversible cleavage complex. Our BGA specifically targeted NCL-positive MCF-7 cells, exhibiting antiproliferative activity, and this suggested that tumor-specific therapeutic aptamers can be developed by using a biased library to screen aptamer candidates for functional targets. Finally, we utilized DM1, which has a synergistic interaction with TOP1 inhibitors, as a conjugated drug. BGA-DM1 exerted an anticancer effect 20-fold stronger than free DM1 and even 10-fold stronger than AS1411 (NCL aptamer)-DM1, highlighting our approach to develop synergistic ApDCs. Therefore, we anticipate that our library might be utilized for the identification of aptamers with effector functions. Furthermore, by employing such aptamers and appropriate drugs, synergistic ApDCs can be developed for targeted cancer therapy in a manner distinct from how ADCs exhibit additional therapeutic efficacy.

Cytotoxic effects of aptamer-doxorubicin conjugates in an ovarian cancer cell line.

Despite medical advances in treatment strategies over the past 30-years, epithelial ovarian cancer (EOC) continues to be defined by poor patient survival rates and aggressive, drug resistant relapse. Traditional approaches to cancer chemotherapy are typically limited by severe off-target effects on healthy tissue and aggressive drug-resistant recurrence. Recent shifts towards targeted therapies offer the possibility of circumventing the obstacles experienced by these treatments. While antibodies are the pioneering agents in such targeted therapies, several intrinsic characteristics of antibodies limits their clinical translation and efficacy. In contrast, oligonucleotide chemical antibodies, known as aptamers, are ideal for this application given their small size and lack of immunogenicity. This study explored the efficacy of a DNA aptamer, designed to target a well-established cancer biomarker, EpCAM, to deliver a chemotherapeutic drug. The results from this study support evidence that EpCAM aptamers can bind to epithelial ovarian cancer; and offers a valid alternative as a targeting ligand with tuneable specificity and sensitivity. It also supports the growing body of evidence that aptamers show great potential for application-specific, post-SELEX engineering through rational modifications. Through in vitro assays, these aptamers demonstrated cytotoxicity in both monolayer and tumoursphere assays, as well as in tumourigenic enriching assays. Further experimentation based on the results achieved in this project might aid in the development of novel cancer therapeutics and guide the novel designs of drugs for targeted drug delivery.