RNA aptamer-mediated RNA nanotechnology for potential treatment of cardiopulmonary diseases

Abstract

Ribonucleic acid (RNA) aptamers are single-stranded RNAs that bind to target proteins or other molecules with high specificity and affinity, modulating biological functions through distinct mechanisms. These aptamers can act n as antagonists to block pathological interactions, agonists to activate signaling pathways, or delivery vehicles for therapeutic cargos such as siRNAs and miRNAs. The advances in RNA nanotechnology further enhances the versatility of RNA aptamers, offering scalable platforms for engineering. In this review, we have summarized recent developments in RNA aptamer-mediated RNA nanotechnology and provide an overview of its potential in treating cardiovascular and respiratory disorders, including atherosclerosis, acute coronary syndromes, heart failure, lung cancer, pulmonary hypertension, asthma, chronic obstructive pulmonary disease (COPD), acute lung injury, viral respiratory infections, and pulmonary fibrosis. By integrating aptamer technologies with innovative delivery systems, RNA aptamers hold the potential to revolutionize the treatment landscape for cardiopulmonary diseases.

1. Introduction

Current drug development increasingly focuses on the identification of novel protein targets implicated in various diseases, as well as the exploration of innovative strategies to effectively interfere with these targets and their corresponding biological processes [1]. Recent advancements have driven a shift from traditional small molecules to complex agents such as peptides, antibodies, adeno-associated viruses, and most recently RNA-based therapeutics [2]. Among these, RNA aptamers have emerged as promising candidates demonstrating high specificity, substantial stability, low immunogenicity, and capabilities for mass production and rapid modification [3,4]. In 2004, FDA approved pegaptanib as the first RNA aptamer designed to selectively target vascular endothelial growth factor (VEGF) for the treatment of neovascular age-related macular degeneration (AMD) [5]. In 2023, avacincaptag pegol, another RNA aptamer, received approval for treating geographic atrophy (GA) secondary to AMD [6]. RNA aptamers offer advantages over traditional therapeutic agents, particularly in their potential for precise molecular targeting in disease management, thereby presenting new opportunities for precision medicine [4].

Cardiovascular and pulmonary diseases are significant health burdens and major contributors to global morbidity and mortality [7]. Conditions like ischemic heart disease, chronic obstructive pulmonary disease (COPD), and lung cancer pose great challenges to healthcare providers due to the limitations of current therapies including limited efficacy or non-specific targets [8–10]. The precise targeting ability of RNA aptamers towards pathological molecules or cell types holds promise to address these disorders while minimizing the side effects [11]. This review explores the potential of RNA aptamers as therapeutics for heart and lung diseases, discussing recent research, clinical applications, and translational challenges. We also highlight how RNA aptamers could improve efficacy and safety of treatments, providing insights into their role in revolutionizing cardiovascular and respiratory disease management.

1.1. Selection of RNA aptamers

RNA aptamers are single-stranded RNA oligonucleotides (20 – 80 nucleotides) that fold into unique three-dimensional structures, enabling selectively binding to certain molecule targets with high affinity and specificity [11,12]. The selection of RNA aptamers is achieved using Systematic Evolution of Ligands by Exponential Enrichment (SELEX), an interative method firstly introduced in 1990, which involves multiple cycles of binding, separation, and amplification to isolate aptamers with the highest binding affinity for their targets (Fig. 1) [4,13,14]. The traditional SELEX process starts by generating a large library containg up to 10¹⁵ random sequence oligonucleotides [4]. These oligonucleotides are subsequently incubated with a target molecule, allowing for the isolation of those exhibiting adequate affinity towards the target [4]. The bound sequences are then separated, reverse-transcribed into complementary DNA (cDNA), amplified using polymerase chain reaction (PCR), and transcribed back into RNA to create an enriched pool for subsequent rounds. This cycle of binding, separation, and amplification is repeated 6 – 15 times to progressively enhance the specificity and affinity of the selected aptamers [4,15]. To further refine the selection, negative selection steps are often included, where the RNA pool is exposed to non-target molecules to remove sequences that exhibit non-specific binding [16].

Fig. 1.

The SELEX Process for RNA Aptamer Selection. A schematic illustration of the SELEX process for RNA aptamer selection. A random RNA library undergoes iterative cycles of binding, separation, and amplification to enrich for sequences with the highest binding affinity and specificity to the target molecule (created by Figdraw).

Traditionally, obtaining high-quality aptamers requires over one month, but recent advancements in SELEX technology have significantly improved the efficiency of aptamer selection [15,17]. High-throughput sequencing and computational analysis has accelerated the identification of high-affinity aptamers from large libraries [15,18]. Other innovations, such as magnetic bead SELEX and capillary electrophoresis SELEX, have streamlined binding and separation, reducing the number of cycles required [19]. Additionally, technologies like cell SELEX and in vivo SELEX have expanded identification and isolation of aptamers within more complex biological contexts, such as whole cells or living organisms, enhancing the relevance of selected aptamers for real-world environment [15]. Together, these advancements not only optimize the conventional SELEX procedure but also extend the clinical application of RNA aptamers, making them more stable and suitable for diagnostic and therapeutic potentials [15,20].

1.2. Mechanism of action of RNA aptamers

RNA aptamers offer a therapeutic strategy by directly targeting the proteins involved in the pathological changes (Fig. 2) [21]. As single-strainded RNA molecules, these aptamers fold into unique three-dimensional structures, which enable them to interact with diverse targets including receptors, ligands, and enzymes [16]. The antagonist function of the RNA aptamer is induced by its binding to either a receptor or ligand to block their interactions, thereby switching off the corresponding downstream signaling [16]. This inhibitory capability has made antagonistic RNA aptamers the predominant focus in clinical trials [21]. On the other hand, aptamers can also serve as agonists by inducing receptor dimerization to trigger subsequent signaling cascades, or transform and stabilize the receptor into an active conformation [22]. These processes mimic or enhance the natural interaction between the intrinsic ligands and receptors, potentiating protective proteins and offering therapeutic benefits in various diseases [16].

Fig. 2.

Mechanisms and Applications of RNA Aptamers. A schematic illustration demonstrating the versatility of RNA aptamers in therapeutic and nanotechnological applications. RNA aptamers can act as antagonists by blocking receptor-ligand interactions or as agonists by activating signaling pathways through receptor dimerization (A). They can also be engineered as chimeras by fusing with functional nucleic acids, such as siRNAs or miRNAs, to enable receptor-specific targeting and precise gene regulation (B). Additionally, RNA aptamers can be integrated into nucleic acid nanoparticles (NANPs), creating multifunctional platforms for targeted therapy, diagnostics, and real-time monitoring (C) (created by Figdraw).

RNA apatmers can be further fused with other nucleic acid-based molecules, such as small interfering RNAs (siRNAs), small activating RNAs (saRNAs), small hairpin RNAs (shRNA), microRNAs (miRNAs), or miRNA inhibitors (anti-miRs), to form RNA aptamer chimeras [23,24]. For example, aptamer-siRNA chimeras enable targeted gene silencing, while anti-miR chimeras correct aberrant miRNA activity. These chimeras provide a target specific method for post-transcriptional gene manipulation, combining the high specificity of aptamers with the regulatory capabilities of nucleic acid therapeutics. Additionally, chimeric RNA aptamers can simultaneously deliver therapeutic cargos an antagonize the pathogenic receptors, functioning as a dual-function system for better efficacy [16]. However, challenges remains particularly in achieving intracellular delievery and tackling endosomal escape [25].

RNA aptamers can also be intergrated into nucleic acid nanoparticles (NANPs), creating a platforms for the attachment of multifunctional units. For instance, NANPs carrying EGFR-targeting aptamer and anti-miR21modules demonstrated tumor-specific suppression in triple-negative breast cancer models while minimizing side effects [26]. This versatile approach offers researchers scalable tools for precision medicine, as NANPs can be engineered to combine therapeutic, diagnostic, and tracking functionalities [27]. Additionally, incorporating fluorophores into these structures enables real-time monitoring of nanoparticle dynamics, providing insights into their behavior and interaction in vivo [28]. This ability to track and optimize delivery further enhances the potential of NANPs as precision therapeutic tools.

2. RNA nanotechnology for respiratory and cardiovascular applications

Recent advancements in RNA nanotechnology can enhance RNA aptamers for better therapeutic and diagnostic applications. RNA nanotechnology involves the self-assembly of nano-scale architectures composed primarily of RNA, including the scaffolds, targeting ligands, and therapeutics [29–46]. The packaging RNA (pRNA) from the phi29 bacteriophage DNA packaging motor has been used as building blocks to construct lung aptamer-guided RNA complex with controllable sizes and shapes. The development of stable phi29 pRNA 3WJ motifs has facilitated the construction of multivalent nanoparticles characterized by enhanced chemical and thermal stability [32]. These RNA nanoparticles are anionic polymers allowing for the construction of homogeneous nanoparticles with defined size, structure, and stoichiometry [31–33,47–51]. They are also capable of harboring other motifs while retaining their functionalities [26,31,38,39,52,53]. Recent developments include more stable RNA nanoparticles, such as multi-branched architectures, spheres, dendrimers, micelles, and prisms, with high stability [31,48,52,54–59].

RNA nanotechnology has shown great promise as an aptamer-guided targeted delivery platform with enhanced tumor retention and minimal accumulation in vital organs, showing immense promise in lung cancer therapy [29,31–39,47,60–67]. Favorable pharmacokinetic profiles of RNA nanoparticles have been demonstrated, and their controllable physical characteristics were investigated for optimal tumor targeting and limited organ accumulation [68–79]. The rubber-like property of RNA architectures facilitates enhanced vessel extravasation and renal excretion, contributing to stronger tumor targeting and less non-specific accumulation compared to rigid nanoparticles [80–83]. The stretchable and shrinkable nature of RNA nanoarchitectures allows for the construction of elegant RNA nanostructures, such as squares, pentagons, and cubes, with multiple extension and relaxation repeats while remaining stable. This dynamic and elastic nature allows RNA nanoparticles to squeeze through leaky tumor vasculature, enhancing their permeability and retention effects (EPR) [46,84]. These properties enable RNA aptamer and RNA nanoparticles to target diseased lung cells spontaneously, in addition to active targeting via ligand/receptor interactions [26,52,55,85,86].

RNA aptamers and RNA nanoparticles also possess quick body clearance, rapid renal excretion, low liver and spleen accumulation, thus minimal toxicity, which offers potential solutions for respiratory and cardiovascular diseases beyond tumors. RNA nanoparticles avoid entrapment in the liver, spleen, and other vital organs via bypassing the mononuclear phagocytic system (MPS) recognition) since the 10–20 nm RNA nanoparticles are below the size cutoff of macrophage engulfment [29,87,88]. The negative charge of RNA nanoparticles creates a repulsion from negatively charged cell membranes, preventing nonspecific binding, further reducing off-target effects [89–91]. Meanwhile, RNA nanoparticles equipped with targeting ligands such as aptamers or chemical moieties demonstrate specific cell-binding capabilities, further enhancing therapeutic efficacy while minimizing side effects[30,33,47,48,92–102].

RNA nanotechnology also enables the functionalization of exosomes for targeted delivery. Exosomes, 30–150 nm vesicles of endocytic origin [103–106], naturally mediate intercellular communication and can cross biological barriers, such as the blood-brain barrier, making them excellent candidates for drug delivery [107–111]. However, their lack of inherent targeting specificity has limited their therapeutic utility [103,107,112]. RNA nanoparticles derived from the stable phi29 pRNA three-way junction (3WJ) motif address this challenge [55,86,113]. By conjugating hydrophobic moieties, such as cholesterol, RNA nanoparticles anchor to exosome membranes, displaying RNA aptamers on the surface for cell-specific targeting [55]. This approach allows engineered exosomes to deliver high payloads of siRNAs, anti-miRs, or other therapeutic agents directly to disease sites. The pRNA-3WJ nanoparticles used for ligand display are homogeneous in size, structure, and stoichiometry [30,32,47,52,58,59,88,114]; can be synthesized chemically in large quantities and self-assembled [39,88]; are thermodynamically [32,115,116] and chemically [33,117] stable, non-toxic [38], non-immunogenic [118], and display a favorable biodistribution and PK/PD profile [31–33,47,92], making them ideal for scalable therapeutic applications. These advancements establish RNA-functionalized exosomes as a promising tool for precision medicine, offering safer and more effective therapeutic delivery options.

Overall, the integration of RNA aptamer technology with RNA nanotechnology presents a promising future for the development of innovative and effective treatments for respiratory and cardiovascular diseases, advancing the fields of precision medicine and nanomedicine.

3. RNA aptamer in the heart diseases

3.1. Atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the arterial wall, highly associated with an increased risk of myocardial infarction and stroke [119]. It initiates with vascular injury and endothelial dysfunction, followed by deposition of lipids and formation of plaques in arterial walls [120]. The rupture of these plaques often leads to thrombosis and eventually death [120]. Standard interventions such as statins function by competitively inhibiting 3-hydroxy-3-methyl glutaryl coenzyme reductase A, the rate-limiting enzyme in cholesterol biosynthesis, effectively reducing low-density lipoprotein cholesterol levels [121]. However, the systemic administration of statins may also impact the muscular metabolism, leading to an increased the risk of myopathy or even rhabdomyolysis, highlighting the need for novel therapeutic strategies specifically targeting the vasculature [122,123].

Aptamers have offered a unique approach by targeting the von Willebrand factor (vWF), a critical glycoprotein involved in plaque formation and thrombosis [124,125]. ARC1779 is a DNA/RNA hybrid anticoagulant aptamer engineered to bind to the A-1 domain of vWF, preventing its interaction with platelet glycoprotein Ib and subsequently inhibiting the risk of thromboembolic events [126]. This aptamer influences multiple steps of the thrombus growth cascade, and is particular effective under the intravascular shear forces in the arterial-side circulation, making it a better choice over traditional antiplatelet agents [126]. A recent Phase II study highlighted the ability of ARC1779 to reduce thromboembolism while also noting potential side effects of bleeding and anemia [126]. However, the study was prematurely terminated due to financial issues [125]. Further, several new aptamers, incuding BT200 and DTRI-031, were developed to inhibit vWF. BT200, a next-generation polyethylene glycol-coated (PEGylated) aptamer, exhibits better tolerability, bioavailability, and longer half-life compared with ARC1779 [127,128]. DTRI-031, another optimized vWF-specific RNA aptamer under Phase I study, has been shown to effectively prevent thrombosis and promote recanalization of occluded vessels by inhibiting platelet adhesion [124]. Importantly, antidotes have been developed to rapidly reverse the activity of BT200 and DTRI-031, thereby enhancing its safety for clinical practice [124,129]. These advancements in targeting vWF could significantly improve the current anti-atherosclerotic medications, particularly for individuals resistant to statins or experiencing severe adverse reactions.

Furthermore, a new RNA aptamer was reported to inhibit proprotein convertase subtilisin/lexin type 9 (PCSK9), a protein binds to low-desity lipoprotein receptors (LDLRs) and induces LDLR degradation, increasing plasma LDL levels and causing hypercholesterolemia [130]. This aptamer prevents PCSK9 from binding to LDL receptors, effectively lowering plasma cholesterol levels and demonstrating its therapeutic promise for treating hypercholesterolemia [130]. Aptamers targeting adhesion molecules like α(v)β(3) integrin and P-selectin have shown effectiveness in reducing cell adhesion under shear stress, a mechanism that could also prevent leukocyte-endothelial interactions during atherosclerosis progression [131]. Similarly, high-affinity RNA aptamers targeting activated protein C (APC), a key regulator of the coagulation cascade, inhibit its protease activity, providing a novel approach to managing thrombosis in atherosclerosis [132].

The pathological process of atherosclerosis is involed with multiple cell types, particularly endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and monocytes/macrophages [133]. The disease initiates with endothelial injury and dysfunction, which subsequently stimulates VSMC proliferation and migration [134]. Recent advancements in RNA aptamer technology offer targeted interventions for these cellular mechanisms. For example, Apt14, an RNA aptamer generated by cell SELEX, has been demonstrated to selectively internalize into VSMCs, effectively inhibiting their proliferation and migration, and affecting the cellular processes driving atherosclerosis [135,136]. Additionally, Apt14 was shown to facilitate endothelial healing and inhibit selective pathways responsible for neointimal hyperplasia in a swine iliofemoral injury model [137,138]. The use of a perfusion catheter for localized delivery further enhances the potential of Apt14 as a precise and cell-specific therapeutic agent, offering a safer and more effective approach for managing vascular diseases [136]. Furthermore, R39, a species cross-reactive, 2′F-modified RNA aptamer, targets endothelial cells and can deliver small interfering RNAs (siRNAs), such as siVEGFR2 to silence genes critical in vascular dysfunction [139]. This blood-brain barrier-permeable aptamer shows promise for atherosclerosis and stroke interventions, with potential applications from preclinical mouse models to human therapy [139].

3.2. Acute coronary syndromes

Acute coronary syndromes (ACS) are characterized by the rupture of atherosclerotic plaques and subsequent blockage of coronary arteries, leading to conditions like unstable angina or myocardial infarction [140]. This severe stage of coronary artery disease necessitates immediate medical intervention to rescue heart function [141]. Essential interventions include promptly percutaneous coronary intervention (PCI) and the deployment of drug-eluting stents (DES) to restore coronary blood flow and minimize myocardial damage [142]. Within the coagulation cascade, Factor IX (FIX) activates and interacts with Factor VIII (FVIII) to form the tenase complex [143]. This complex is crucial for activating Factor X (FX), leading to a series of enzymatic reactions that ultimately result in clot formation [144]. The integration of RNA aptamer technology offers a promising therapeutic approach to reduce the thrombosis complications following stent implantation. Notably, the RNA-aptamer-based FIXa inhibitor pegnivacogin effectively suppressed thrombin and reduced platelet activity in patients with ACS, offering reliable anticoagulation strategy during cardiac catheterization [145–147]. The development of anivamersen as a specific antidote for pegnivacogin further enhances its safety profile by enabling rapid reversal of anticoagulant effects if necessary [146,148]. REG1, an anticoagulation system consisting of pegnivacogin and its controlling anivamersen, went through Phase I and IIa clinical trial [149]. However, the Phase IIb trial was prematurely terminated due to three severe allergic reactions in the REG1 arms, later attributed to pre-existing anti-PEG antibodies instead of the RNA aptamer itself [150]. Additionally, a Phase III study was halted early due to allergy concerns but demonstrated a comparable therapeutic effect to bivalirudin in reducing ischemic events [151]. Although RNA aptamer technology like REG1 system shows promise to manage coagulation in ACS patients, challenges in immune response must be carefully addressed for safe clinical use. DTRI-178, a novel anticoagulant aptamer targeting factor IXa, demonstrated efficacy in preventing thrombosis without impairing hemostasis in a piglet model of extracorporeal membrane oxygenation (ECMO), offering a potential advancement in managing coagulation complexities in ACS [152]. Apt02, an RNA aptamer targeting integrin alpha V (ITGAV), holds promise in reducing cardiac fibrosis after myocardial infarction by blocking stromal cell activation and fibrotic tissue formation [153,154]. Moreover, a kallikrein-targeting RNA aptamer, developed using SELEX, has been shown to inhibit kallikrein and prekallikrein, extending clotting times and reducing bradykinin release, offering another avenue for innovative anticoagulant therapies in cardiovascular diseases [155].

3.3. Heart failure

Heart failure, a complex clinical syndrome arising from cardiac disorders such as ACS or hypertension, leads to impaired pumping function and compromised circulatory efficiency [156]. Traditional treatments, including beta-blockers, angiotension-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and diuretics, along with angiotensin receptor-neprilysin inhibitors (ARNIs), aim to manage symptoms and prevent disease progression [157]. In the meantime, emerging therapies aim to target the molecular mechanisms of heart failure more precisely [158]. Among these, RNA aptamers are being explored for their ability to modulate proteins involved in cardiac remodeling, fibrosis, and inflammation, offering a more personalized treatment approach [158]. These aptamers intervene at the molecular level, alleviating symptoms, improving cardiac function, and potentially altering disease progression [158].

For instance, the chymase-specific RNA aptamer, HA28, has been shown to significantly improve cardiac function and reduce mortality rates in the heart failure following myocardial infarction by suppressing chymase gene expression and activity [159]. Similarly, it has been shown that an RNA aptamer reversed cardiac dysfunction and fibrosis in a mouse model of pressure overload by blocking osteopontin (OPN) signaling, and reducing gene expression related to extracellular matrix and hypertrophy [160]. Additionally, Gint4.T-MP aptamer-peptide chimera presents a unique strategy by incorporating an internalizing aptamer to specifically deliver therapeutic anti-PDGFRβ peptides to cardiac cells, thereby addressing defects in the L-type calcium channel activity [161]. A GRK2-specific RNA aptamer inhibits kinase activity by binding to the ATP pocket, showing potential for mitigating heart failure progression driven by GRK2 upregulation[162]. Similarly, an RNA aptamer-based sensor targeting inflammatory marker lipocalin-2 could enable real-time monitoring of cardiovascular stress, improving diagnostic precision and timely intervention[163]. Furthermore, RNA aptamers targeting the monomeric form of C-reactive protein (mCRP), a biomarker strongly implicated in inflammation and cardiovascular disease, provide a promising avenue for distinguishing and mitigating inflammation-driven cardiac damage [164,165]. These aptamers not only enable sensitive detection of mCRP but also offer potential for therapeutic modulation of inflammation in heart failure. Together, these developments underscore the potential of RNA aptamers as revolutionary tools in the treatment of heart failure, offering approaches that go beyond conventional therapies.

4. RNA aptamer in the lung disease

4.1. Lung cancer

Lung cancer is the leading cause of cancer-related deaths, accounting for around 127,000 deaths in the US in 2023 [166]. Non-small cell lung cancer (NSCLC) represents the predominant subtype, encompassing adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma [167]. NSCLC often involves mutations within the tyrosine kinase domain (TKD) of the epidermal growth factor receptor (EGFR) gene [168]. Although tyrosine kinase inhibitors (TKIs) are initially effective, resistance typically develops within 12–18 months due to secondary (T790M) and tertiary (C797S) mutations, along with other oncogenes such as MET or mutations in downstream genes like KRAS [169]. In response to these challenges, RNA aptamers have emerged as a novel therapeutic approach that provides precise targeting of molecular pathways involved in tumor growth and resistance mechanisms.

Recently, an anti-wildtype (WT) epidermal growth factor receptor (EGFR) aptamer named MinE07 was developed to target the most common oncogenic mutations in the tyrosine kinase domain in NSCLC [169]. CL4 is an RNA aptamer that targets EGFR with high specificity, effectively inhibiting signaling pathways and inducing apoptosis in cancer cells resistant to standard EGFR inhibitors [170]. CL4 has been shown to enhance anti-cancer effectivity, particularly in NSCLC, when combined with cetuximab [170]. Further, a modified CL-4 aptamer, CL-4RNV616, has been developed. It displayed enhanced stability in human serum and higher affinity to EGFR, also showed antiproliferative effect against breast cancer and glioblastoma cells [171].

To enhance the ability to target intracellular protein, RNA aptamers can be delivered into cells through an adenoviral system. For example, A-p50 aptamer delivered via an adenoviral system effectively suppresses NF-kappaB in NSCLC A549 cells, reducing doxorubicin resistance by targeting cell proliferation, apoptosis, and angiogenesis pathways, offering a novel strategy for enhancing chemotherapy efficacy [172].

In addition to directly bind to receptor and inhibit its biological functions, RNA aptamer can also be used as a carrier to deliver other small RNAs including siRNA and miRNA to the target cells. Tyrosine kinase receptor Axl is highly expressed on lung cancer cells. Laoni M. et al. conjugated an innovative RNA aptamer GL21.T, targeting the Axl receptor, with miRNA-212. GL21.T interacts Axl on the cell surface of lung cancer cells and deliver miRNA-212 into cancer cells through GL21. T/Axl internalization. Increases in mRNA-212 levels by the RNA aptamer/miRNA delivery system enhanced TRAIL therapy in NSCLC [173]. This chimera sensitizes cancer cells by decreasing the anti-apoptotic protein PED/PEA-15, enhancing apoptosis, and significantly reducing tumor proliferation and metastasis [173].

RA16, a 2′-F RNA aptamer identified by in vivo SELEX, exhibits strong binding affinity to NCI-H460 lung cancer cells and inhibits their proliferation. When conjugated with epirubicin, RA16-epirubicin nanoparticles showed enhanced anticancer effect both in vitro and in mouse xenograft models [174]. The subsequent study revealed that both a synthetic version of RA16 (syn-RA16) and a truncated form (S3) effectively target and suppress NCI-H460 cell growth, demonstrating their potential for NSCLC treatment and highlighting their promising tumor-targeting capabilities in vivo [175].

RNA aptamers targeting cancer stem cell markers such as CD133 have shown enhanced tumor penetration and retention compared to monoclonal antibodies. These aptamers, including the smallest described 15-nucleotide RNA aptamer, specifically recognize the AC133 epitope and exhibit superior performance in 3-D tumor sphere models, laying the groundwork for stem cell-targeted therapeutics in NSCLC [176]. Similarly, aptamers designed to target epithelial cellular adhesion molecule (EpCAM), overexpressed in various carcinomas, demonstrate high affinity for EpCAM dimers, which are more physiologically relevant than monomers [177]. For example, RNA aptamers also enable precise modulation of signaling pathways involved in tumor progression. Phototoxic RNA aptamers provide innovative application. These aptamers selectively bind cancer-specific glycan-peptide markers on epithelial cells and deliver photodynamic therapy agents such as chlorin e6. Upon light activation, these aptamers exhibit over 500-fold enhanced toxicity against cancer cells [178]. Aptamers targeting MAP kinase (MAPK) pathways selectively inhibit ERK2 phosphorylation without affecting related kinases like JNK or p38. This specificity offers a pathway-focused approach to cancer therapy, reducing off-target effects [179].

RNA aptamers are being developed as tools for diagnostics and imaging in NSCLC. Aptamers targeting HER-2, overexpressed in some lung cancers, have been successfully isolated using cell-SELEX. These aptamers specifically bind HER-2 and show potential as reagents for early detection and therapeutic targeting of HER-2-positive cancers [180]. In addition to diagnostics, aptamers targeting transcription factors, such as beta-catenin, modulate oncogenic transcription and alternative splicing in colon cancer cells, and similar approaches could be applied to NSCLC [181–186].

To further improve the structural stability and delivery efficiency of RNA nanoparticles, 3WJ-based multi-functional RNA nanoparticles were developed and further exemplified the advancements in targeted therapy, which can efficiently and specifically deliver siRNA or miRNA into targeted cell types [187]. The rubber-like deformation property of 3WJ RNA nanoparticles further enhance tumor targeting by allowing them to squeeze through leaky tumor vessels, thereby improving the enhanced permeability and retention effect [84]. Additionally, this rubbery property enables the nanoparticles to be rapidly metabolized by the kidneys and excreted through urine, resulting in lower toxicity [84]. Moreover, the 3WJ nanoparticles have been shown to enhance the solubility of hydrophobic antitumor drugs, such as camptothecin, for targeted delivery in cancer treatment [39]. Yang et al. have designed a 3WJ nanoparticles containing a 3WJ core, EGFR aptamer, and siRNA for KRAS^G12C to target and silence KRAS^G12C in lung cancer cells, resulting in the attenuation of MAPK pathway, a key driver of lung cancer cell proliferation [188]. Recently, four-way junction (4WJ) RNA nanoparticles have been developed to conjugate with the anticancer drug SN38, demonstrating highly efficient cancer inhibition in orthotopic lung metastases of colon cancer [189]. These 4WJ nanoparticles exhibited rapid renal excretion, quick body clearanceand, and minimal toxicity or immunogenicity, making them a promising and safe option for drug delivery in future clinical applications [190].

Aptamer technology for treating lung cancer involves bispecific aptamers, and combination with other therapies in order to increase specificity and decrease toxicity. Innovations like lipid nanoparticles will improve their stability and precise delivery. Integrating aptamer technology into standard lung cancer treatments could provide more personalized and effective options [191].

4.2. Pulmonary hypertension

Pulmonary hypertension (PH) is a chronic, progressive disease defined as elevated mean pulmonary artery pressure over 25 mmHg [192]. This condition involves significant functional and structural changes within the pulmonary vasculature, primarily driven by aberrant states of pulmonary arterial SMCs (PASMCs) and ECs (PAECs) [192]. Upon cellular injury, PAECs shift to an anti-apoptotic phenotype, resulting in the thickening and constriction of distal arterioles, which in turn increases pulmonary vascular resistance and arterial pressure [193]. This process together with continuous proliferation of PASMCs during hypoxia leads to irreversible changes like hyperplasia and plexiform lesions, ultimately leading to right heart failure and premature death [194].

Recent research has focused on molecular targets within the PH pathogenesis, notably endothelin-1 (ET-1), a potent vasoconstrictor that is upregulated in PH patients [195]. ET-1, primarily released from ECs, exerts its effects through endothelin A (ETA) and endothelin B (ETB) receptors.[195]. Activation of ETA receptors induces vasoconstriction and proliferative responses, exacerbating the vascular structural changes [195]. Current therapeutic strategies targeting ET-1 involve the use of endothelin receptor antagonists (ERAs) such as bosentan, ambrisentan, and macitentan [196]. These agents block ET-1 from binding to its receptors, thus mitigating vasoconstriction and vascular remodeling, and have demonstrated efficacy in improving the functional capacity and quality of life in PH patients [196].

Although no RNA aptamers are currently available for targeting ET-1 or its pathways in PH, advancements in DNA aptamer research offer valuable insights. For instance, the synthetic DNA aptamer BC007, designed for in vivo neutralization of pathogenic autoantibodies directed against G-protein coupled receptors (GPCR-AABs), specifically ETA-AABs for PH patients, which are also implicated in cardiovascular diseases like dilated cardiomyopathy (DCM) [197]. BC007 has demonstrated efficacy in vitro and in vivo, effectively neutralizing these autoantibodies, producing the therapeutic potentials observed with immunoadsorption, also potentially offering a more accessible and cost-effective alternative [197–199]. This approach directly targets autoantibody-mediated mechanisms, possibly enhancing current therapy for PH by addressing both acute and chronic aspects of the disease at the molecular levels [198,199]. Another DNA-based aptamer NX1975 has been developed to target PDGF-B chain, a downstream mediator of ET-1 effects, and has been shown to reduce vascular thickening and right ventricular hypertrophy in models of PH [200,201]. Furthermore, activation of ET1 is induced by the transforming growth factor β (TGF-β)/Smad3 pathway, which contributes to smooth muscle cell proliferation and altered apoptosis, highlighting another target for therapeutic intervention [202].

While current treatments focus on DNA aptamers, developing RNA aptamers could open new therapeutic avenues, potentially offering advantages in specificity and stability, and manage this complex disease more effectively.

4.3. Asthma and allergy

Asthma and allergy are heterogeneous diseases that is characterized by hyper-immune responses, airway inflammation, and hyperresponsiveness. Diverse inflammatory mediators, including interleukin-4 (IL-4), IL-5, and IL-13, contribute to the pathogenesis of asthma. These cytokines are crucial components of adaptive immune responses and promote excessive production of immunoglobulin E (IgE) antibodies by T-helper 2 (Th2) lymphocytes, thereby exacerbating allergic responses. Exposure to allergens initiates IgE-mediated activation of mast cells, resulting in the release of mediators such as histamine and leukotrienes, which induce immediate airway constriction and subsequent inflammatory responses. Chronic inflammation induces airway remodeling, resulting in progressive and frequently airway obstruction, which underscores the persistent severity of asthma symptoms.

Theophylline was historically used as a bronchodilator for treating bronchial asthma and chronic obstructive pulmonary disease (COPD) [203]. However, it has a narrow therapeutic range which necessitates precise monitoring to avoid toxicity, particularly due to its potential to induce ventricular fibrillation [203]. The structure of theophylline is similar with caffeine and theobromine, which often complicates its measurement in serum using conventional methods like chromatography and immunoassays [203]. RNA aptamers have been utilized as highly specific molecular tools for the monitoring of theophylline levels, ensuring safety in asthma management [204].

However, the use of theophylline has markedly declined in recent years, overtaken by more effective and safer treatments like β2-agonists and inhaled corticosteroids (ICSs), which provide superior bronchodilatory and anti-inflammatory benefits [204]. Despite this shift, therapeutic focus has transitioned from monitoring therapeutic range to inhibiting the inflammatory mechanisms underlying asthma. For example, mepolizumab, reslizumab, and dupilumab are monoclonal antibodies respectively targeting IL-5, IL-5 receptor α-subunit, and IL-4 receptor α-subunit, showing efficacy and safety in patients with severe eosinophilic asthma [205,206]. In addition, unnatural cyclic peptides that bind to IL-5 were identified through SELEX from mRNA-displayed peptide libraries, highlighting the capability of RNA technology to develop novel therapeutic agents that target specific inflammatory pathways [207]. Furthermore, recent advancements have led to the development of the DNA aptamer CCS13, acting as a CD200R1 agonist to suppress inflammatory responses in murine models of skin graft rejection and house-dust-mite-induced allergic airway inflammation, further illustrating the therapeutic potential of aptamers in regulating complex immune responses [208]. As both cyclic peptides and DNA aptamers significantly impact treatments in allergic diseases, RNA aptamers should continuely be developed to improve, with the potential to introduce novel therapeutic approaches for asthma management by modulating inflammation rather than merely managing bronchodilation.

4.4. COPD

COPD, recognized as the third leading cause of death globally, is a progressive respiratory disease marked by obstructed airflow in the lungs, largely due to chronic inflammation caused by long-term cigarette smoke exposure [209]. Transforming growth factor-β1 (TGF-β1), a multifunctional cytokine, together with activation of the Smad pathway in the airways have been implicated in COPD pathogenesis [210]. Recently, an innovative DNA aptamer was developed to specifically target TGF-β signaling [211]. This aptamer was reported to effectively counteract the negative effects of TGF-β on cystic fibrosis transmembrane conductance regulator (CFTR), an essential protein for mucociliary clearance (MCC) [211]. Research also revealed that TGF-β and cigarette smoke remarkably increased miR-145–5p levels, leading to the suppression and dysfunction of CFTR [211]. Combination a TGF-β-neutralizing DNA aptamer with miR-145–5p antagonism has been demonstrated to restore CFTR functionality [211]. These findings revealed the mechanisms underlying cigarette smoke-induced COPD, and showed a bright future for RNA aptamer therapy targeting TGF-β/CFTR pathways.

4.5. Acute lung injury

Acute lung injury (ALI) is a critical condition triggered by lung-directly or indirectly inflammatory insults such as pseudomonas infection, sepsis, or trauma, possibly progressing to acute respiratory distress syndrome (ARDS), which results in significant morbidity and mortality [212–214]. Its pathogenesis involves a complex cascade of events, including the activation of alveolar macrophages by Toll-like receptor (TLR) and NOD-like receptor (NLR) pathways, leading to the recruitment of neutrophils, and the release of proinflammatory cytokines and neutrophil extracellular traps (NETs) [212]. These inflammatory responses disrupt the alveolar-capillary barrier, causing pulmonary edema and impaired gas exchange [212]. Treatment is primarily supportive, focusing on addressing the underlying cause of the disease [212].

Recent research has highlighted the role of Angiopoietin-2 (Ang2) in exacerbating pulmonary inflammation in ALI [215]. Elevated circulating levels of Ang2 have been observed in patients with ALI, underscoring its contributory role in the pathogenesis of pulmonary inflammation [215]. As an antagonist of the receptor tyrosine kinase Tie2, Ang2 counteracts the protective effects of Angiopoietin-1 (Ang1), thereby promoting vascular inflammation [215]. In the mouse model of endotoxin-induced ALI, elevated Ang2 levels did not notably affect vascular leak or mortality but significantly increased lung inflammation. An innovative therapeutic strategy involves using selective Ang2 binding RNA aptamer to inhibit its activity [216]. In the lipopolysaccharide (LPS)-induced acute lung injury (ALI) models, treatments with Ang2-neutralizing RNA aptamer resulted in a significant reduction in total cell counts and protein concentrations in bronchoalveolar lavage fluid, indicating decreased inflammation and vascular permeability [217]. These findings suggest that RNA aptamer therapy targeting Ang2 could be beneficial to improve clinical outcomes of ALI or ARDS patients.

Another promising therapy for ALI is HBA7, an RNA aptamer selectively binds extracellular histones [218,219]. Extracellular histones are released and act as damage-associated molecular patterns (DAMPs) that cause cellular toxicity and promote inflammation in ALI [220]. HBA7 demonstrated significant efficacy in mitigating neutrophil infiltration, interstitial edema, and barrier disruption induced by histone protein inhalation, thereby reducing lung inflammation and enhancing alveolar permeability [218]. Furthermore, this aptamer exhibited therapeutic in the model of lung injury cansed by wood smoke particulate matter [218]. These findings demonstrate the potential of HBA7 as an RNA-based therapeutic for ALI, especially a targeted delivery through inhalation to the lungs.

RNA aptamers targeting the transcription factor NF-κB can modulate inflammation and immune responses by binding to its DNA-binding domain, preventing interaction with duplex DNA and reducing proinflammatory signaling [221,222]. Aptamers against proinflammatory cytokines such as IL-32 and IL-10 receptors have shown promise in mitigating inflammation. IL-32-targeting aptamers reduced TNF-α induction in lung carcinoma cells, while IL-10 receptor-blocking aptamers inhibited tumor growth in murine models, potentially modulate immune responses in ALI [223,224]. Moreover, RNA aptamers designed to bind teichoic acid on Staphylococcus aureus cell walls exhibited strong affinity, demonstrating utility in bacterial infection-triggered ALI [225]. These aptamers could enhance pathogen detection and neutralize bacterial virulence factors, reducing infection-induced inflammation. These advances underscore the versatility of RNA aptamers in addressing key inflammatory and pathogenic mechanisms in ALI.

4.6. Viral respiratory infections

Respiratory infections, especially in the upper respiratory tract, are typically caused by viral pathogens such as severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) or influenza A virus (IAV) [226,227]. The clinical severity of these infections ranges widely, from asymptomatic or mild disease of the upper airways to severe illnesses like acute respiratory failure and ARDS [226].

During the SARS-CoV-1 outbreak, the therapeutic potential of RNA aptamers has already been demonstrated. ES15 as an RNA aptamer against the SARS-CoV-1 NTPase/Helicase (nsP10) effectively inhibited the helicase’s DNA unwinding activity by up to 85 % with an IC₅₀ value of 1.2 nM, highlighting its potential as an anti-SARS‑CoV agent [228]. Similarly, during COVID-19 pandemic, high-binding-affinity aptamers was developed targeting the receptor-binding domain (RBD) of the spike glycoprotein of SARS‑CoV‑2 [229]. Using an ACE2 competition-based selection strategy, the optimized aptamers CoV2-RBD-1C and CoV2-RBD-4C exhibited dissociation constants (Kd) of 5.8 nM and 19.9 nM, respectively, indicating their potential applicability in the treatment of SARS-CoV-2 infections [229]. Additionally, a 2′-fluoro protected RNA aptamer that also binds to the RBD of SARS-CoV-2 spike protein was developed. The aptamer prevented the interaction between virus and ACE2 receptor and neutralizing viral entry [230]. A trimerized version of this aptamer enhanced binding affinity to the low picomolar range and efficiently blocked viral infection in vitro [230]. Furthermore, a newly identified RNA aptamer, Apta-1, has been shown to exhibit therapeutic benefits against coronaviral infection-induced lung injury and systemic inflammatory responses [231]. In a murine model subjected to intranasal administration of murine hepatitis virus 1 (MHV-1), treatment with Apta-1 reduced viral titers, prevented hemorrhage, and decreased chemokine levels [231]. This aptamer showed significant anti-inflammatory effects and improved clinical outcomes, underscoring its potential in the treatment of SARS-CoV-2 infections and the attenuation of related complications [231]. These findings revealed the potential of RNA aptamer therapy in treating coronaviral infections [232].

Current RNA aptamers for influenza virus infection treatment can directly target viral structural proteins [233]. For example, the RNA aptamer P30–10–16 specifically binds to the hemagglutinin (HA) region of the human IAV subtype H3N2 (A/Panama/2007/1999), showing remarkable specificity and affinity [234]. Another aptamer, D-26, targets the HA protein of the pandemic H1N1 influenza virus (A/California/07/2009) and inhibits HA-mediated membrane fusion and glycan interactions with significant affinity and specificity [235]. Additionally, RNA aptamers have also been developed for avian influenza. The RNA aptamer HAS15–5, which was selected for its affinity with the HA1 domain of the H5 avian influenza virus, showed significant antiviral activity in hemagglutination inhibition assays [236]. Similarly, the HA12–16 RNA aptamer, which specifically target the glycosylated receptor-binding domain of HA, effectively inhibited viral infection in host cells by neutralizing the receptor-binding site [237]. Another aptamer, 8–3, exhibits high affinity to hemagglutinins of highly pathogenic avian influenza viruses H5N1 and H7N7, disrupting HA-glycan interactions and preventing viral attachment to host cells [238]. These examples showed the versatility of RNA aptamers as antiviral therapies against diverse influenza virus strains, presenting a promising strategy in disease control.

Moreover, RNA aptamers targeting the IL-6R have been developed to mitigate cytokine storm associated with severe viral infections, such as COVID-19. Specifically, an RNA aptamer exhibiting high affinity for IL-6R (Kd = 20 nM) was identified, which facilitated the internalization of IL-6R. This aptamer demonstrated the capability to deliver therapeutic cargos into IL-6R-expressing cells, as evidenced by the delivery of fluorescently labeled streptavidin [239]. Another RNA aptamer, IBR, inhibited the interaction between IL-6 and IL-6R, demonstrating potential for reducing systemic inflammation [240]. Additionally, RNA aptamers targeting Toll-like receptor 3 (TLR3) ectodomains offer a unique approach to modulate the antiviral immune response, with high-affinity aptamers binding TLR3 without agonist or antagonist effects [241]. Overall, RNA aptamers offer promising solutions for viral respiratory infections by targeting viral proteins, inflammatory cytokines, and immune signaling pathways.

4.7. Lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by abnormal fibroblast proliferation and excessive extracellular matrix (ECM) production, leading to decreased lung function and eventually respiratory failure [242–244]. At present, IPF remains incurable but two antifibrotic agents, nintedanib and pirfenidone, have demonstrated efficacy in slowing down the progression of the disease [245]. Nintedanib, a receptor antagonist for multiple tyrosine kinases, mitigates inflammation and fibrosis by targeting PDGF, VEGF, and fibroblast growth factor receptors [246]. Pirfenidone primarily exerts its antifibrotic effect through inhibition of TGF-β, collagen synthesis, ECM production, and fibroblast proliferation [247].

Recent studies of RNA aptamers present therapeutic opportunities for pulmonary fibrosis. Specifically, one aptamer has been identified to target lung fibroblasts, the critical factor in the progression of IPF [248]. This aptamer was selected using cell SELEX, and has demonstrated a high affinity for IPF-derived lung fibroblast cells (Kd = 70 nM) [248]. When conjugated with monomethyl auristatin F, this aptamer effectively inhibits fibroblast proliferation without affecting non-targeted epithelial cells, indicating its potential for precise and targeted drug delivery in the treatment of lung fibrosis [248].

TGF-β drives fibrosis through its receptor complexes, with the type III receptor (TGFBR3) acting as a coreceptor to enhance ligand binding. TGFBR3 downregulation promotes fibroblast differentiation via Smad-dependent and Smad-independent pathways[249]. RNA aptamers targeting TGFBR3, developed using the TECS-SELEX method, exhibit high affinity (Kd = 1 nM) and effectively compete with TGF-β for receptor binding, which demonstrates therapeutic potential for lung fibrosis [250].

Additionally, it has been demonstrated that chronic oxidative stress drives fibrosis progression, with increased Reactive Oxygen Species (ROS) levels detected in patients with IPF [251]. Cells generate reactive oxygen species (ROS) through the activity of NADPH oxidases (NOX), encompassing NOX1–5 as well as dual oxidases DUOX1 and 2 [252]. An ongoing APFIBROX project focuses on developing 2′-Fluoro-Pyrimidine RNA aptamers targeting DUOX1 through whole cell SELEX to inhibit TGF-β1-induced myofibroblastic differentiation, and test their efficacy in a bleomycin-induced pulmonary fibrosis mouse model [253].

5. Challenges and future directions

Although RNA aptamers offer tremendous potential for treating heart and lung diseases, their clinical translation faces several challenges that must be addressed to facilitate widespread implementation (Table 1). A major barrier is to ensure the stability of aptamers in the bloodstream, as unmodified aptamers are susceptible to rapid degradation by nucleases and subsequent excretion through the kidneys. Chemical modifications, such as the incorporation of 2′-fluoro and O-methyl groups, and PEGylation, have markedly improved the stability and bioavailability of aptamers [254]. However, PEGylation has raised concern due to its immunogenicity, as evidenced by the presence of anti-PEG antibodies in a propotion of populations [255,256]. PEG-specific antibodies, either pre-existing or induced by PEGylated therapeutics, form immune complexes that accelerate clearance, reduce efficacy, and induce hypersensitive reactions [256,257]. Pegnivacogin, a PEGylated RNA aptamer against FIXa, was prematurely terminated from phase 3 clinical trials due to severve allergic reactions in patients with anti-PEG antibodies [258]. To address this concern, the measurement of anti-PEG antibodies should be a standardized procedure before administration of any PEGylated nanomedicine. Establishing a set of standards like samples containing different PEG epitopes would ensure consistency and accuracy of assays across laboratories [257]. Future efforts should also focus on designing PEG alternatives like zwitterionic and hydrophilic polymers, which mimic its properties while minimizing the immune reactions. Studies have suggested changing the terminal group of PEG from methoxyl to hydroxyl can reduce the immunogenicity [259]. Innovative encapsulation strategies such as lipid nanoparticles or exosome-based delivery can provide a physical barrier against the immune recognition, further mitigating the issue [257]. The development of biologically inert polymers and better delivery systems, including biodegradable platforms for controlled release, will be essential for addressing these challenges and realizing the full therapeutic potential of RNA aptamers.

Table 1.

RNA aptamers with potential applications in cardiovascular and respiratory disease treatment.

Aptamer	Target	Related disease	Affinity (Kd)	Reference
Apt02	Integrin alpha v	Cardiac fibrosis after myocardial infarction	359 nM	[153]
ARC1779	A1 domain of von Willebrand Factor (vWF)	Atherosclerosis	2 nM	[267]
BT200	A1 domain of vWF	Atherosclerosis	5 nM	[128]
DTRI–031	A1 domain of vWF	Atherosclerosis	11 nM	[124]
PBR	PCSK9	Atherosclerosis	73 nM	[130]
Apt14	Vascular smooth muscle cell (VSMC)	Atherosclerosis	1.705 ± 0.783 nM	[137]
R39	Microvascular endothelial cell	Atherosclerosis	3.4 ± 1.2 nM	[139]
α(v)β(3) aptamer clone 17.16	Endothelial integrin alpha-v beta–3	Atherosclerosis	30 nM	[131]
APC 99	Human Activated Protein C	Atherosclerosis	137 nM	[132]
REG1	Factor IX	Acute coronary syndrome	N/A	[149]
DTRI–178	Factor IXa	Acute coronary syndrome	N/A	[152]
HA28	Chymase	Heart failure	0.17 nM	[159]
OPN aptamer	Osteopontin (OPN)	Heart failure	N/A	[160]
Gint4.T	Platelet-derived growth factor receptor β (PDGFRβ)	Heart failure	9.6 nM	[268]
GRK2-C13.28	G protein-coupled receptor kinase 2 (GRK2)	Heart failure	1.2 nM	[162]
mLcn2-Oligo 569	Mouse Lipocalin–2 (mLcn2)	Heart failure	340 nM	[163]
MinE07	Epidermal growth factor receptor (EGFR)	Lung cancer	1–2 nM	[169]
CL4	EGFR	Lung cancer	38 nM	[170]
CL–4RNV616	EGFR	Lung cancer	18.24 nM	[171]
A-p50	NF-κB	Lung cancer	N/A	[172]
GL21.T	Axl tyrosine kinase receptor	Lung cancer	12 nM	[269]
RA16	NCl-H460	Lung cancer	24.75 ± 2.28 nM	[174]
CD133-A15	CD133	Lung cancer	83.2 ± 82.9 nM	[176]
CD133-B19	CD133	Lung cancer	145.1 ± 75.4 nM	[176]
EP23	Epithelial cell adhesion molecule (EpCAM)	Lung cancer	39.89 ± 3.37 nM	[177]
cAMP	cAMP	Lung cancer	10 μM	[186]
26A-t	Basic Fibroblast Growth Factor (bFGF)	Lung cancer, pulmonary fibrosis, ischemic heart disease	190 pM	[185]
RNA 14–16	p68 oncogenic helicase	Lung cancer	13.8 nM	[184]
ERK1/ ERK2 (Family II)	Extracellular Regulated Kinase 1 and 2 (ERK 1 and ERK2)	Lung cancer	N/A	[179]
5TRG2	Epithelial cancer cell	Lung cancer	18.6 nM	[178]
S6	HER2	Lung cancer	94.6 nM	[180]
A30	HER3	Lung cancer	45 nM	[183]
h-Ras–1	Human Ras protein (h-Ras)	Lung cancer	200 nM	[270]
Beta-catenin	Arm 1–12 of B-catenin	Lung cancer	5 nM	[182]
N71yc16	Protein tyrosine phosphatase (PTPase)	Lung cancer	18 nM	[181]
mTCT8–4	Theophylline	Asthma	100 nM	[271]
11–1.41	Angiopoietin–2	Acute lung injury	3–10 nM	[216]
HBA7	Extracellular histones	Acute lung injury	N/A	[218]
Staphylococcus aureus RNA Aptamer	Staphylococcus aureus	Acute lung injury	N/A	[225]
AIR–3A	IL–6R	Inflammtion	20 nM	[239]
C-Reactive Protein Monomer	Monomeric C-Reactive Protein (CRP)	Inflammtion	187.7 nM	[164]
CRP1–1	CRP	Inflammtion	2.25 nM	[165]
R5A1	IL–10R	Inflammtion	12 nM	[224]
#411-J	IL–10R	Inflammtion	18 nM	[272]
AC3–3	IL–32	Inflammtion	78 nM	[223]
Aptamer 3	NF-kB	Inflammtion	1 nM	[222]
D1	NF-kB p65	Inflammtion	149 nM	[221]
R1	NF-kB p65	Inflammtion	11 nM	[221]
R2	NF-kB p65	Inflammtion	25 nM	[221]
SARS-CoV nucleocapsid protein	SARS-CoV nucleocapsid (N) protein	SARS-CoV	1.65 nM	[273]
ES15–1	SARS CoV NTPase/Helicase	SARS-CoV	1.2 nM	[228]
CoV2-RBD-1C	SARS-CoV–2 RBD ACE2	SARS-CoV–2	5.8 nM	[229]
CoV2-RBD-4C	SARS-CoV–2 RBD ACE2	SARS-CoV–2	19.9 nM	[229]
RBD-PB6	SARS-CoV–2 spike protein	SARS-CoV–2	18 nM	[230]
Apta–1	heparin-binding motif (Exosite II) on thrombin	SARS-CoV–2	N/A	[231]
TLR3-ECD (Family–1)	Toll-like receptor 3 Ectodomain	Influenza infection	2.1 nM	[241]
P30–10–16	Human Influenza A virus H3N2 Hemagglutinin (HA)	Influenza infection	0.188 nM	[234]
D–26	H1N1 HA	Influenza infection	200 nM	[235]
HA Clone B	H3N2 HA	Influenza infection	115 pM	[274]
HAS15–5	H5 Avian Influenza Virus HA	Influenza infection	N/A	[236]
HA12–16	gHA1	Influenza infection	N/A	[237]
8–3	H5N1 HA	Influenza infection	25 nM	[238]
A20	Influenza B HA	Influenza infection	720 pM	[275]
A065	LL97A cells	Lung fibrosis	70 nM	[248]
A07	TGFBR3	Lung fibrosis	2.47 nM	[250]

Another significant challenge is the precise and effective delivery of aptamers to target tissues, such as cardiac or lung tissues, while minimizing off-target effects [260]. Up-to-date platforms such as 3WJ or 4WJ RNA nanoparticles, lipid or exosome-based systems are still being explored to enhance the precision of aptamer therapies. For instance, functionalizing exosomes with aptamers offers an approach for direct delivery to diseased tissus, reducing systemic toxicity. The integration of RNA aptamers into existing therapies will enhance the effects of conventional medications and mitigate their adverse effects. For example, the concurrent administration of aptamers with other anticoagulant agents has shown improved effectiveness in controlling coagulation processes during cardiac surgical interventions [261]. Combining aptamers with cutting-edge therapies, such as immune checkpoint inhibitors or CRISPR gene-editing tools, could induce synergistic effects and expand their therapeutic applications. Furthermore, incorporating aptamers into theranostic platforms could offer dual therapeutic and diagnostic functions, enabling real-time monitoring of treatment responses.

Despite numerous studies reporting improved aptamers and advanced constructs (e.g. magnetic, gold, silicon, or chitosan nanoparticles), few have progressed to clinical trials. One possible reason is the predominant focus on cell-based studies and delays in transitioning to animal models, including non-human primates. We recommend early collaboration between chemists, biomedical engineers, and biomedical researchers to initiate in vivo studies promptly and generate robust animal data that can support clinical translation. Furthermore, establishing large-scale patient-specific aptamer libraries could expedite the identification of aptamers tailored to individual genetic profiles or disease biomarkers, thereby promoting personalized medicine. Advances in computational modelling, including artificial intelligence and machine learning can accelerate the design and optimization of aptamer specificity and efficacy [262]. For example, advanced machine learning models could rapidly predict high-affinity aptamer candidates de novo, bypassing traditional experimental methods.

While both RNA and DNA aptamers demonstrated high specificity and versatility across various applications, key differences impact their suitability for specific uses [263]. RNA aptamers typically form more intricate three-dimensional structures than DNA aptamers, allowing for greater conformational diversity and binding capabilities [264]. However, this diversity often comes at the expense of stability, as RNA is more prone to degradation due to the presence of a 2’-hydroxyl group that makes it susceptible to hydrolysis and nuclease attack [265]. DNA aptamers, in contrast, exhibit greater native stability, with in vitro half-lives that far exceed those of unmodified RNA aptamers [266]. Despite these limitations, chemical modifications such as 2’-fluoro substitutions can enhance RNA aptamer stability to equal or even surpass DNA aptamers [254]. The choice between RNA and DNA aptamers often depends on the structure requirement and stability need of the application.

From a broader perspective, the aptamer field also faces significant challenges in scalability, manufacturing consistency, and regulatory approval. Although aptamers are synthetically produced, it is still a complex task to achieve clinical-grade production with minimal batch-to-batch variability while meeting the regulatory standards. Addressing these issues is essential to attract industry interest, which currently lags behind other RNA-based therapeutics like siRNAs or mRNAs. Synthetic biology approaches could further enhance scalability by enabling precise and consistent aptamer production in engineered cell-free systems. By overcoming these challenges, RNA aptamers could transform the treatment of cardiovascular and respiratory diseases, providing innovative solutions for unmet clinical needs.

Data Availability

No data was used for the research described in the article.