Aptamers: Current Applications in Leukemia Diagnostics and Therapeutics

Background

Aptamers are chemically synthesized, single-stranded oligonucleotides (DNA, RNA or XNA) that fold into three-dimensional shapes, enabling high-affinity and specific noncovalent binding to target molecules.^54–59 Some of these targets include metals, small organic molecules, nanoparticles, chemical linkers, peptides, proteins, fluorophores, toxins, drugs, viruses, bacteria, and whole cells (see Fig. 1). ^{59 – 68}

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FIG. 1.

A schematic illustrating the diverse targets of oligonucleotide aptamers (proteins, ⁶⁰ ions, ⁶¹ small molecules, ⁶² cells, ⁶³ viruses, ⁶⁴ and bacteria, ⁶⁵ accompanied by relevant examples of binding interactions, highlighting some of their potential applications. Of note, the ion panel shows the interaction of one g-quadruplex region of an aptamer sequence that is stabilized by a metal cation. In addition, in the cell detection panel, the yellow symbol indicates a fluorophore (donor), and the black symbol indicates a quencher/FRET acceptor. “Signal OFF (FRET)” reflects donor quenching when fluorophore and quencher are proximal; “Signal ON” reflects decreased FRET and increased fluorescence upon target binding that separates the pair. Image recreated from original using BioRender under the Creative Commons Attribution License from. ⁶⁶ The ion-binding aptamer panel was adapted from Zhou et al. Chemical Reviews, 2017; Figure 2E. ⁶¹

Aptamers have applications in a variety of fields including biological imaging,^69,70 food safety, and medicine.^71,72 In recent years, they have been studied as a tool for the diagnosis^{52,66,67,69,73–75} and therapeutic treatment of a variety of cancers.^10,49,76,77 Aptamers are ideal vectors for cancer therapies, as they can bind with specific biomarkers associated with cancer cells and have the means to induce cytotoxic effects without affecting healthy cells in the process. ⁷⁷ With respect to therapeutics, aptamers have been studied in a variety of contexts, including as a delivery method for chemotherapeutic drugs or as a standalone therapeutic through preclinical studies and clinical trials.^{13,49,52,76–79} Pegaptanib (Macugen) is a selective RNA aptamer that targets VEGF165 and was the first aptamer drug to receive regulatory approval. It also became the first anti-VEGF therapy approved for neovascular age-related macular degeneration. Clinical studies showed that it significantly slowed vision loss compared with sham injections and maintained an acceptable safety profile over as long as 3 years of treatment.^80–82

Antibodies and aptamers operate in a similar fashion in that they share the same targets and have high affinity/specificity for those targets.^{14,54–56,67,83} Despite the similarity in mechanism of action, aptamers have been shown to have several advantages over antibodies, including low production cost,^10,49,51,68 ease of production and modification,^{5,10,49,52,67} better tissue penetration due to their small size (8–25 kDa compared with ∼150 kDa for antibodies),^{49,66,68,76,84–86} and little to no immunogenicity or toxicity. ⁸⁷ Of note, aptamers that have been successful through the clinical trial process tend to be RNA aptamers that are heavily modified and therefore have increased costs of production associated with them. The requirement for modifications is a practical bottleneck of bench to bedside translation for aptamer research. Because aptamers are oligonucleotides, they do not generate an immune response like antibodies (or modified oligonucleotides) and therefore may not elicit the unwanted side effects associated with an activated immune response.^{5,10,51,52,67,68,73,76,88} In addition, aptamer selection may be performed rapidly in vitro, whereas antibodies are typically produced in vivo, which is a much longer process. Antibody production often requires the use of animals or cells, which limits target classes for antibodies to those that are nontoxic, unlike aptamers that can essentially be selected to any target that can form hydrogen bonds.^67,73,76,89 Not only is in vitro chemical synthesis a faster process, but it also allows for better reproducibility^51,67 and the synthesis may be automated, which increases efficiency of the process. ⁷³ In vitro selection also provides more flexibility in the experimental conditions, allowing the aptamers to function under nonphysiological conditions. ⁹⁰ Moreover, aptamers are also more stable at higher temperatures. When the temperature is raised, they may denature but will return to their native state when the temperature is lowered, allowing a return of function. ⁹⁰ Conversely, antibodies will completely lose function when denatured, which is irreversible.^{10,52,66,68,79}

Despite the advantages of aptamers, there are some associated challenges with their use. Unmodified aptamers are hard to translate into the clinic mainly because they do not last long enough in the body and are cleared too quickly. Because they are nucleic acids, aptamers are susceptible to nuclease degradation and clearance, which results in a short half-life in vivo ⁷⁹ and can eliminate activity. Their small size, while helping tissue penetration, also means they are rapidly filtered out by the kidneys, sometimes within minutes after intravenous dosing, so there is often not enough time to achieve a meaningful therapeutic effect. Aptamers also have a relatively simple chemistry since they are made from only four nucleotides, which can limit how well they interact with certain targets and increases the need for engineered formats or chemical modifications. Although modifications can improve stability and extend circulation, aptamers typically still have much shorter half-lives than monoclonal antibodies, so stability remains the main hurdle.^{49,54–56,79} One notable modification is the replacement of the 2′-hydroxyl of the ribose sugars with a fluoro, O-methyl, or amino group.^76,79 Another known modification for this issue is polyethylene glycol (PEG)ylation, which has been used in different studies.^{49,68,71,72,91} The PEG modification has also been seen to increase the molecular weight of the aptamer in question to counteract rapid renal secretion.^68,79 Although this may be helpful for the concern of renal secretion, increasing the molecular weight may cause problems with target binding ⁷⁹ and may initiate an immune response. ⁹² PEGylation is a well-established drug delivery strategy that is widely used to improve pharmacokinetics and pharmacodynamics across many types of therapeutics, including aptamers. More than 30 PEGylated drugs are already in clinical use, and many others have advanced into clinical trials. ⁹³ However, PEGylation is not risk-free: preexisting anti-PEG antibodies have been linked to acute hypersensitivity reactions after first exposure to pegnivacogin, a PEGylated RNA aptamer, highlighting a clinically important immunological concern that needs to be considered when using PEG in therapeutic design. ⁹⁴ Finally, even though clinical tumor resistance to aptamer therapies has not been clearly documented, it should still be considered when designing and evaluating new aptamer-based treatments. ⁹⁵

As an alternative to chemical modification, Spiegelmers may be used to prevent nuclease degradation l-ribonucleic acid aptamers (l-RNA aptamer, trade name Spiegelmers) are aptamers in mirror image form, adopting an l-sugar backbone as opposed to the traditional d-sugar backbone conformation. ⁷⁹ They will not be degraded, as nucleases are only able to recognize nucleic acids that contain sugars in the dextro configuration.^13,79

Selection

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a method through which oligonucleotide aptamers can be selected from DNA or RNA libraries for specific targets. Figure 2 illustrates the SELEX process. This evolutionary process consists of four main steps: incubation, partitioning, recovery, and amplification via PCR. During the incubation step, a strategically designed partially randomized oligonucleotide library is incubated with the target of interest, such as proteins, whole cells, or other targets. ⁵⁰ For a comprehensive discussion of SELEX considerations, see DeRosa et al. ⁹⁶ During the partition stage, bound oligonucleotide sequences are separated from the unbound sequences through a variety of heterogenous methods such as filtration, affinity chromatography, and magnetic bead-based separation, as well as homogeneous partitioning techniques such as capillary electrophoresis.^43,97,98 Bound sequences are then eluted from the target and subsequently amplified by PCR. DNA sequences may be directly amplified, while RNA sequences must be reverse transcribed to generate cDNA before amplification. ⁵⁰ PCR products then undergo subsequent rounds of SELEX to enhance the affinity of the sequences for the target. ⁵⁰ There are a few different methods that can be used to improve selectivity. This includes the use of competitors, increasing washing times to remove oligonucleotides that do not bind as well and decreasing the amount of the target (molecules, proteins, cells, etc.). ⁷⁶ Traditionally, oligonucleotides would then be cloned and sequenced. However, next-generation sequencing can now be used to bypass the cloning step. ⁹⁹ In general, DNA aptamer selections are becoming more and more common, but RNA selections are also possible. In the case of RNA selections, an extra step is involved where the PCR product postamplification must be reverse transcribed before moving to the newest selection round.

With this general procedure in mind, different SELEX methods vary slightly. For example, when selecting Spiegelmers, the incubation stage cannot utilize a native target, but rather one of the selection against the mirror image of the target. ⁹⁸ Other changes have led to new SELEX methods such as protein-based SELEX, cell SELEX, and in vivo SELEX, to name a few. ⁵⁰ Protein-based SELEX is a common method that uses proteins as the aptamer target, mainly purified recombinant protein biomarkers. ⁵⁰ However, this method is limited because it may not be used when the identity of the protein biomarker of interest is unknown, insoluble, or has a complex structure. ⁵⁰ It has also been seen that aptamers selected through this method sometimes cannot recognize their protein targets in vivo because the proteins adopt a different conformation in vivo than in vitro, or the protein may be posttranslationally modified, unlike the recombinant protein that was used as the target. ⁵

The cell SELEX method (Fig. 3A) utilizes live cells that express the biomarker of interest.^50,66,91 The most common target of cell-SELEX is proteins on or embedded in the surface of the cell. However, it is possible that aptamers in cell-SELEX could also bind to other targets such as lipids or sugars. The advantage of cell-SELEX is that the target is in its natural state; for example, proteins will be in their native conformational state on the cell surface. ⁶⁶ Because proteins do not need to be purified for this method, the protein target does not need to be known,^50,66 and it can therefore be used for the identification of biomarkers in cancer cells. ⁹¹ Cell SELEX involves the general steps of SELEX as shown in Figure 2; commonly, a counterselection step that follows the core SELEX process is added prior to the incubation of the library with target. This counterselection step helps ensure aptamers will only bind with cells containing the target of interest.^50,66,91 During this process, cells should be viable and nonmalignant to minimize nonspecific binding and selection of off-target aptamers; if not, nonspecific binding aptamers may become more prevalent. ⁵⁰ Additional SELEX variations, including in vivo SELEX and related approaches, are illustrated in Figure 3B and C.

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FIG. 2.

Schematic representation of the Systematic Evolution of Ligands by Exponential Enrichment process steps (left) and available partitioning methods (right). Image recreated from original using BioRender under the Creative Commons Attribution License from. ⁶⁶

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FIG. 3.

Panel A: Schematic diagram illustrating Cell-SELEX cycle. Panel B: Aptamers are easily chemically modified, and so they allow for the conjugation of molecules such as siRNAs (1), drugs (2), and nanoparticles (3) via common functional group addition to the aptamer terminals (amine, carboxylic acid, thiol, etc.). ¹⁰⁰ Typically, the efficacy of therapeutic aptamers and their conjugates are assessed in cellular and/or rodent models (Panel C). Image recreated from original using BioRender under the Creative Commons Attribution License from. ¹⁰¹ In this figure, the linker is labeled in purple, while the nanoparticle is shown in yellow.

In vivo SELEX uses live animals to generate specific aptamers in vivo and does not require the counter-selection step, as cell-SELEX does. ⁵⁰ This method has been seen in a study conducted by Mi et al. ¹⁰² whereby the group used in vivo selection to generate RNA aptamers selected to target colorectal cancer metastases; the aptamers were shown to accumulate selectively in liver tumors while sparing normal tissues. The molecular target was identified as p68 RNA helicase (DDX5), with binding confirmed at a dissociation constant (K_d) of 14 nM.

Leukemia SELEX examples

Earnest et al. ¹⁰³ developed aptamers against AML cells associated with oncogene MLL-AF9, which is expressed in human blood stem/progenitor cells. Three candidate sequences were identified, from which one lead aptamer (KGE02) was selected based on binding affinity and specificity. Flow cytometry determined that the lead aptamer, modified with a 5′ fluorescein tag, had an approximate binding constant of 37.5 ± 2.5 nM to AML cells. To further confirm the lead sequence binding, fluorescence and confocal microscopy were performed to understand the localization of the cognate biomarker for KGE02. Fluorescein-tagged KGE02 was incubated with the cells and showed distinct localization in the membrane of the AML cells, which necessitates further analysis for drug-aptamer conjugates.

In another study, Amano et al. ¹⁰⁴ demonstrated that RNA aptamers were developed to bind with the Runt domain (RD) of the AML1 protein, an essential transcription factor involved in the development of hematopoietic cells. Genetic variations of AML1 can lead to abnormal hemocytopoiesis and immunodeficiency, often detected in human leukemia. This study suggested that RNA aptamers, by specifically binding to the AML1 RD, could offer new insights into the treatment of AML1-related diseases.

Aptamer KH1C12 was compared with two other control cell lines (K562 and NB4) using flow cytometry and exhibited significant selectivity to the target AML cell line (HL-60) and was able to recognize the target cells within a complex sample of normal bone marrow. ⁷⁵ They also identified another aptamer, KHG11, which can bind with significant selectivity to target AML cell lines such as HL-60 and human promyelocytic leukemia cells. ⁷⁵

Using the Cell-SELEX approach, Shangguan et al.^105,106 identified the DNA aptamer Sgc8c-7, which targets protein tyrosine kinase 7 (PTK7). PTK7 is highly expressed on the T-ALL cell line CCRF–CEM, suggesting that Sgc8c-7 could be useful for developing more precise and effective targeted approaches for ALL.

Aptamers for leukemia diagnosis

There are many emerging studies on applying aptamer technology to proof-of-concept systems for diagnosing leukemia. Yu et al. ¹⁰⁷ have developed a simple and rapid electrochemical aptamer cytosensor for the direct detection of CML K562 cells based on a specific aptamer and a biotin-conjugated concanavalin A (bio-ConA) detection probe. Recoveries of 79.6%–93.3% were obtained when testing to detect K562 cells in human blood samples, which suggests that the biosensor could be a potential tool for CML K562 cell detection in biological sample. Although this method has strong potential as a leukemia diagnostic tool, reproducibility is still challenging. ¹⁰⁸

Recently, a study used the thickness-shear mode acoustics method (TSM) and single-molecule force spectroscopy (SMFS) to study the interactions between DNA aptamers (sgc8c) specific to the PTK7, which is known to be a biomarker of leukemia lymphoblasts (MOLT-4). ⁷⁴ In this study, the frequency changes concerning MOLT-4 cell lines are more significant in comparison with Jurkat cells and the U266 (control) cell lines. In this method, the sensing layer formed by thiolated Sgc8c aptamers is very sensitive to both low concentrations of MOLT-4 and Jurkat cells. Therefore, the TSM method allowed the development of a highly sensitive, label-free biosensor for the detection of leukemia cells. The SMFS method also proved the high selectivity of the Sgc8c aptamers to the PTK7 receptors. ⁷⁴

Yang et al. ¹⁰⁹ used the cell-SELEX approach and worked toward selecting DNA aptamers that target NB4 AML, a human promyelocytic leukemia cell line. The target protein of the K19 aptamer was identified as the sialic-acid-binding immunoglobulin-like lectins (Siglec-5). The aptamer was able to successfully detect small amounts of AML cells in human bone marrow samples. In this study, the researchers employed a combination of flow cytometry, fluorescence imaging, and mass spectrometry to evaluate aptamer binding and identify target proteins on AML cells. Flow cytometry was the primary quantitative tool used to assess aptamer binding affinity and specificity across leukemic cell lines and primary bone marrow samples by measuring fluorescence intensity after aptamer staining. Fluorescence microscopy provided visual confirmation of aptamer localization on the cell surface, supporting the flow cytometry data. To identify the molecular target of the lead aptamer (K19), mass spectrometry was conducted following affinity purification, which led to the discovery of Siglec-5 as the aptamer’s cognate receptor. These complementary techniques enabled both the functional characterization and molecular validation of the aptamer-based diagnostic strategy. ¹⁰⁹

A method based on aptamer-modified fluorescent silica nanoparticles (FSNPs) has been developed to detect leukemia cells. Amine-labeled Sgc8 aptamer was conjugated to carboxyl-modified FSNPs, which resulted in a highly sensitive and specific aptamer-modified FSNP system. This was due to the amplified fluorescence signal caused by the high density of FITC fluorophore in the nanoparticles and specific target binding by the aptamer. The study suggested that the Sgc8-FSNPs system may be a good candidate for leukemia diagnosis. ¹¹⁰

Mallikaratchy et al. ¹¹¹ identified the aptamer TD05, which binds to Ramos cells. They chemically modified TD05 to covalently cross-link with its target on Ramos cells to capture and enrich the target receptors using streptavidin-coated magnetic beads. This process was followed by mass spectrometry, which revealed that the target for the TD05 aptamer is membrane-bound immunoglobulin.

Simple colorimetric sensors/assays can eliminate the use of analytical instruments to obtain results and, therefore, are attractive as convenient rapid test methods. Bamrungsap et al. ¹¹² developed aptamer-conjugated magnetic nanoparticles (ACMNPs) to sensitively detect leukemia cells. A PTK7-binding DNA aptamer (sgc8c) was used to recognize CCRF–CEM cells and confirmed specificity with clear sequence controls: sgc8c-ACMNPs gave a strong signal on CCRF–CEM cells, while a control aptamer conjugate (TDO5-ACMNPs) produced little to no signal, and the same control logic was applied using Ramos cells. The assay detects cells by measuring changes in spin–spin relaxation time (T2) without requiring wash steps, and it still worked in mixed samples containing both target and nontarget cells, using a random-sequence DNA–MNP conjugate as an additional negative control. Importantly, they also tested the method in more realistic biological samples such as serum, plasma, and whole blood, which supports its potential for clinical leukemia cell detection.

A recent study investigated the detection of leukemia cells using aptamer-conjugated, gold-coated magnetic nanoparticles on a nitrogen-doped graphene-modified electrode. A thiolated sgc8c aptamer was immobilized on gold-coated magnetic Fe₃O₄ nanoparticles (Apt-GMNPs). Ethidium bromide was intercalated into the stem of the aptamer hairpin, serving as a signal for quantifying leukemia cells. This approach demonstrated high sensitivity, selectivity, robustness, and simplicity for the early detection of leukemia. In addition, this aptasensor was successfully applied to detect leukemia cells in complex samples, such as human blood plasma, without significant interference. ¹¹³

Aptamer-based biotherapy

In the area of biotherapy, aptamers are used as standalone therapeutics to interfere with intracellular systems or signaling to induce apoptosis. ⁴⁹ AS1411 and NOX-A12 can be considered the most notable examples, as they have both shown promising results in clinical trials for the treatment of leukemia. ⁴⁹ AS1411 is a 26-nucleotide, guanine-rich DNA oligonucleotide that has often been described as a nucleolin-targeting aptamer. However, it differs from most aptamers in an important way: AS1411 was not discovered through SELEX but was instead designed to form a G-quadruplex structure. ¹¹⁴ Later studies, including work from the original group, showed that AS1411 enters cells mainly through macropinocytosis and that this uptake does not depend on nucleolin expression. ¹¹⁵ In addition, its uptake can be reduced by nonspecific competitor oligonucleotides, suggesting that internalization is largely nonspecific rather than driven by a specific receptor interaction. ⁶³ Despite these mechanistic limitations, AS1411 advanced to phase II clinical testing in AML (NCT00512083), where it showed a good safety profile and modest clinical activity. Overall, AS1411 may not behave like a classical, target-specific aptamer, but it remains an important early oligonucleotide therapeutic and a clear reminder that rigorous target validation is essential in aptamer-based drug development.

It has been proposed that AS1411 can interact with nuclear factor-κB, which lowers the activity of BCL-2 mRNA and consequently inhibits cancer cell proliferation and induces cell death.^52,78 The phase II clinical trial (NCT00512083) initiated in 2007 tested intravenous injection of AS1411 into 70 patients with AML. ⁷⁹ Although reduced side effects were observed, therapeutic efficacy was evaluated independently through measures of tumor burden reduction and cell viability. ⁴⁹ After the promising initial results, AS1411 was eventually used in a study for metastatic renal cell carcinoma.^76,79 However, this study did not receive the same positive results as the AML study and, therefore, showed that biomarkers in AS1411-responsive tumors are required.^116,117

NOX-A12 is a 45-nucleotide Spiegelmer that has been studied for the therapeutic treatment of CML, which is known to be untreatable with conventional chemotherapeutic methods. ⁴⁹ Because NOX-A12 is a Spiegelmer, it has the advantage of being resistant to nuclease degradation. ¹³ NOX-A12 targets CXCL12, which binds with CXCR4 and CXCR7 in the cell, each of which possesses a role in cancer cell proliferation, metastasis, and motility.^13,118 A preclinical study conducted by Hoellenriegel et al. ¹³ found that NOX-A12 also caused chemosensitization, allowing the cancer to be treated with chemotherapeutic drugs. ¹³ Currently, there are eight clinical trials that investigate this reagent, two of which are ongoing, one started in 2019, and the other in January of 2026 (https://clinicaltrials.gov/search?term=NOX-A12%20). Apoptosis was induced as a consequence of the aptamer-mediated disruption of CXCL12 signaling rather than by a conventional cytotoxic agent. ⁴⁹ NOX-A12 has also undergone phase II clinical trials (NCT01486797) for CLL among other cancers. ¹³ The trial found that when in combination with bendamustine and rituximab, the treatment had success compared with traditional chemotherapy. ⁴⁹

Cell-selective chemotherapeutic delivery

Several studies have been conducted involving the use of aptamers as a mode of delivery for chemotherapeutic drugs, siRNA, miRNA, and shRNA to target cancer cells. ¹¹⁹ Aptamers may be conjugated to cellular delivery systems such as liposomes to enable targeted transport of therapeutics to malignant cells.^51,67 Conversely, therapeutic payloads can be attached to aptamers through covalent linkers or associated noncovalently (e.g., via intercalation), enabling aptamer-guided delivery of small molecules and nucleic acid cargos.^57,67 Upon receptor binding, many aptamer–drug delivery constructs are internalized primarily through receptor-mediated endocytosis and subsequently traffic to endo/lysosomal compartments, enabling intracellular release of the payload. ⁶⁷ When the therapeutic has been internalized, it then begins to induce its effects without damaging surrounding healthy tissue. Kotula et al. ⁷⁷ used a nucleolin aptamer AS1411 as a delivery tool to help a chimeric aptamer enter leukemia cells. This chimera carried a β-arrestin 2–inhibitory aptamer (β-arr2A3) and was tested in leukemia cells from a mouse CML model as well as in human leukemia samples. In these systems, the β-arr2A3 construct interfered with β-arrestin-related signaling and was associated with reduced leukemia cell growth.

Another study carried out by Zhang et al. ¹²⁰ showed the development of a polyvalent aptamer system for the delivery of Dox to leukemia cells through binding with PTK7. Experimentation found that the poly-aptamer approach showed greater binding affinity with the target compared with monovalent aptamer approaches as well as a greater ability to eliminate cancerous cells. Furthermore, Huang et al. ¹²¹ showed that conjugation of Dox to aptamer Sgc8c-7 resulted in highly efficient targeted delivery of Dox to CCRF–CEM cells, with minimum uptake by off-target cells; the results mentioned above highlight the benefits of using aptamers in clinical applications.

Zhao et al. ⁵³ developed an aptamer targeting the CD117 receptor, which is highly expressed on AML cells. In follow-up experiments, they found that the aptamer preferentially bound membrane-associated IgM (mIgM) on the cell surface, suggesting that CD117 expression may be linked—directly or indirectly—to how mIgM is displayed on leukemic cells. Notably, the aptamer did not bind soluble IgM in plasma, which supports the idea that its interaction is specific to cell-surface targets rather than circulating IgM. CD117 is a transmembrane receptor that is highly expressed on leukemia cells in 95% of relapsed AML patients. The anti-CD117 aptamer sequence contained a G-rich core section and G-quadruplex functional structure, with a length of 79 bases. The result of this study showed the aptamer 1-F specifically binds to CD117 as the AML biomarker with high-affinity K_d (4.24 nM) and was able to specifically be internalized into CD117-expressing cells. This aptamer was later conjugated to methotrexate to form a therapeutic Apt-MTX. ⁵³

Other therapeutic methods

Apart from biotherapy and cell-selective chemotherapy, other aptamer-mediated therapies studied include gene therapy, targeted nanomedicine, and immunotherapy. Gene therapy utilizes an aptamer conjugated to different types of RNA (siRNA, miRNA, etc.) to silence oncogenes and consequently cause cell death. ⁴⁹ While there are limited studies in this area as it relates to leukemia, there are studies in relation to other cancers. ¹²²

In targeted nanomedicine, aptamers are conjugated with nanoparticles, which are delivered to target cells to induce cell death. ⁴⁹ As mentioned above, there are cases where nanoparticles have been conjugated with aptamers and other chemotherapeutic drugs for delivery to the cells. For example, Ho and colleagues ¹²³ recently developed a new CRISPR-Cas9 delivery system. This system targets the critical gene interleukin-1 receptor accessory protein (IL1RAP) in human leukemia stem cells (LSCs). They achieved this by using mesenchymal stem cell membrane-coated nanofibril (MSCM-NF) scaffolds loaded with lipidoid-encapsulated Cas9/single guide RNA ribonucleoprotein and the chemokine CXCL12α.352. The MSCM-NF scaffolds effectively facilitated the targeted delivery of Cas9/IL1RAP sgRNA to LSCs, reducing the growth of LSCs and decreasing leukemic burden in a mouse model of AML.

Mei et al. ¹²⁴ employed rolling circle amplification to generate self-assembled DNA nanoflowers (NFs) with a high drug loading capacity of 71.4% w/w. These nanomaterials were designed to overcome multidrug resistance (MDR) in cancer therapy by incorporating cell-targeting aptamers (e.g., KK1B10 for leukemia and Sgc8 for breast cancer) directly into the DNA template. The aptamers enabled selective recognition and internalization into cancer cells, including drug-resistant variants, thereby enhancing doxorubicin retention and minimizing off-target toxicity. NFs delivered doxorubicin into target chemosensitive and MDR cancer cells, preventing drug efflux and enhancing drug retention in MDR cells. Moreover, NF-Dox induced potent cytotoxicity in target cells while minimizing side effects in nontarget cells, effectively circumventing MDR and reducing side effects.

In addition, Douglas et al. ¹²⁵ developed a novel DNA hexahedral barrel nanostructure to control a switch using the conformational change of a nucleic acid aptamer. A DNA aptamer-based lock secures a closed, hexahedral barrel that holds molecular cargo, such as proteins or small molecules. In this design, the barrel is kept closed by an aptamer sequence paired with a partially complementary strand. When the nanorobot encounters its specific antigen (or target), these antigens bind to the aptamer sequences on the locks. As the aptamers bind to the antigens, their conformational changes cause them to dissociate from their partially complementary strands. The dissociation of the aptamer–antigen complex results in the release of the locks, effectively “unlocking” the barrel. Once the locks are removed, the structural constraints that maintain the closed state are lifted, allowing the barrel to open and release its payload. Research has shown that this structural system enhances selectivity for target tumor cells and enables controlled antibody drug release, effectively suppressing the proliferation of leukemia cells.

In 2013, Wu and colleagues ¹²⁶ developed a multifunctional aptamer nano assembly (AptNA) for precise cancer therapy targeting. The assembly involves loading the antisense nucleotide (AS) of MDR-1 onto a specially designed DNA nano assembly (Apt-NA) attached to a specific aptamer. This treatment has proven effective in eliminating drug-resistant myeloid leukemia cells. The aptamer, KK1B10, recognizes the drug-resistant cell line K562/D, and the AptNA is selectively taken up by the cell. MDR-1-AS is then released to combat the cell’s drug resistance through inhibition of P-glycoprotein expression. Experimental findings have confirmed the specific cytotoxic impact of AptNA on the targeted tumor cells.

Aptamer-based immune redirection has also been investigated in other contexts through dual targeting designs; however, the examples discussed here emphasize CAR-T engineering and RNAi-mediated gene silencing.^49,122,127 Leukemia immune-engaging therapies have progressed more in antibody-based formats than in aptamer-based systems. Blinatumomab, a monoclonal antibody that engages T cells, is one example. Clinical trials have demonstrated its ability to treat ALL, showing that malignant cells can be targeted effectively by cytotoxic T cells (MT-103-211). ¹²⁸ A comparison of aptamers used for diagnostic and therapeutic purposes is summarized in Table 2.

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Table 2.

Comparison of Aptamers Used for Diagnostic and Therapeutic Purposes

Aptamer	Application	Target	Drug (if applicable)	Leukemia type	Control aptamer used?	Competitor/blocking used?	Tested on cells?	Tested in vivo?	Ref
1-F-MTX	Diagnosis/Therapy	CD117	MTX	AML	Yes	Yes	Yes	No	53
K19	Diagnosis	Siglec-5	—	AML	Yes	Yes	Yes	No	109
KH1C12	Diagnosis	H60 cells	—	AML	Yes	Yes	Yes	No	75
KHG11	Diagnosis	H60 cells	—	AML	Yes	Yes	Yes	No	75
S30-T1	Therapy	CD33	DOX	AML	NR	NR	Yes	Yes	5
Truncated TD05	Diagnosis	BCR	—	AML	Yes	Yes	Yes	Yes	85
Sgc8c	Diagnosis/therapy	PTK7	Daunorubicin	ALL	NR	NR	Yes	No	74
AS1411	Therapy	Not definitively validated (often described as nucleolin-associated)	—	AML	NR	NR	Yes	Yes	79
Sgc8c-7	Therapy	PTK7	DOX	ALL	Yes	NR	Yes	No	121
Sgc8c-7	Therapy	PTK7	NHC-Au	ALL	Yes	NR	Yes	No	129
β-arr2A3	Therapy	β-arrestin 2	—	CML	Yes	No	Yes	No	77
NOX-A12	Therapy	CXCL12	Combo therapy (bendamustine + fludarabine)	CLL	Yes	NR	Yes	No	13

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia I N-heterocyclic carbene gold; CML, chronic myeloid leukemia; DOX, doxorubicin; MTX, methotrexate; NHC-Au, complex; NR, not reported.