The Modification Strategies for Enhancing the Metabolic Stabilities and Pharmacokinetics of Aptamer Drug Candidates

Abstract

Aptamers are single-stranded DNA or RNA that can mimic the functional properties of monoclonal antibodies. Aptamers have high affinity and specificity for their target molecules, which can make them a promising alternative to therapeutic antibodies or peptide ligands. However, many aptamer drug candidates in clinical development have been discontinued due to suboptimal metabolic stabilities and pharmacokinetics. To address these issues, chemical modification can be used to enhance the metabolic stability and prolong the half-life of aptamer candidates. The chapter reviewed published data regarding the metabolic stability and pharmacokinetics of aptamer drug candidates from preclinical and clinical studies. The benefits and possible shortcomings of current modification strategies used in these aptamers were briefly discussed.

1. Introduction

Aptamers are single-stranded DNA or RNA oligonucleotide-based synthetic molecules that can replicate the functional features of monoclonal antibodies. Nucleic acid aptamers are generally screened from a library of random nucleic acids utilizing systematic evolution of ligands by exponential enrichment (SELEX) technology [1]. To effectively create an aptamer against a given target molecule, the SELEX procedure requires multiple phases. The aptamer and desired target molecule are incubated to initiate the binding affinity process, which is followed by the division of bound and unbound sequences. After that, binding sequences are amplified by PCR, and ssDNA is extracted to start a new cycle [2, 3]. As a result, aptamers have a high affinity and selectivity for a wide range of target molecules, including peptides, proteins, tiny compounds, and even living cells. Because inhibitory aptamers that affect the activity of pathogenic target proteins may be utilized as therapeutic agents directly, they are a viable alternative to therapeutic antibodies or peptide ligands [4]. Aptamers have several benefits over antibodies, including ease of synthesis, reduced time and cost, lesser immunogenicity, greater stability, and superior refold ability. As a result, aptamers are prospective replacements for homologous antibodies. The United States Food and Drug Administration (FDA) granted approval for the initial aptamer drug (Macugen®) to treat wet-form neovascular age-related macular degeneration (AMD) in 2004 [5, 6]. Several therapeutic aptamers are currently undergoing clinical trials in phases II and III.

However, aptamer candidates’ druggability can be considerably impacted by their metabolic stability and pharmacokinetics. Indeed, due to inadequate qualities in these domains, certain aptamers under clinical development have been abandoned. The renal filtration cut-off value is 30–50 kDa. Native nucleic acid aptamers face challenges in vivo due to their susceptibility to nuclease-mediated cleavage and rapid renal filtration, primarily attributed to the abundance of serum nucleases and their relatively low molecular weight (20 kDa). Consequently, these aptamers are rapidly eliminated from the body, leading to a short biological half-life. These are key issues that are impeding the clinical translation of the nucleic acid aptamer. Chemical modification can be utilized to improve the metabolic stability and half-life of aptamer candidates to solve these difficulties. To begin, changes in metabolic stability may increase nucleic acid aptamer formation while increasing nuclease resistance. Second, long-term alterations might permit nucleic acid aptamer boosting molecular weight substantially over the renal filtration cut-off level (Figure 1). The chapter reviewed published data regarding the metabolic stability and pharmacokinetics of aptamer drug candidates from preclinical and clinical studies. The advantages and disadvantages of current modification procedures employed in these aptamers were briefly explored.

Figure 1.

The common strategies in the chemical modifications of nucleic acid aptamers and their purposes.

2. Strategies for enhancing metabolic stability of aptamers

Modification tactics for improving metabolic stability of aptamer drug candidates may be carried out in two ways: pre-SELEX chemical modifications and post-SELEX chemical modifications. Pre-SELEX alterations mostly entail chemical changes that are critical for the aptamer’s functions [6]. Nucleobase modifications and genetic alphabet extension are typical examples. The goal of adding these new chemical moieties or bases to aptamers is to improve their functionality and allow them to engage with additional targets. These changes immediately affect the three-dimensional structure of aptamers [7]. Aptamer activity may be fully eliminated by adding these chemical groups following SELEX. As a result, pre-SELEX alteration is the most effective strategy to minimize activity loss. The fundamental disadvantage of this method is that the alteration might impair the nucleotide’s capacity to act as a substrate for DNA or RNA polymerase [8]. During solid-phase chemical synthesis, changes at multiple sites are added to preselected aptamers in post-SELEX techniques for the best performances such as high affinity, high stability, and high specificity. Because aptamer affinity/specificity and function are structure-dependent, post-SELEX alteration may modify the intrinsic characteristics and folding structures of the original aptamers, compromising binding affinity [9]. As a result, alterations must be properly tailored to the intended functions [10]. Unfortunately, general guidelines do not exist for all aptamers, and tedious evaluation/optimization is frequently required [11]. The following are some of the most prevalent nucleic acid aptamer modifications:

2.1 Nucleobases modifications

The research on SELEX using unnatural nucleobases has seen rapid growth in recent years, with two main categories of efforts: (1) creating unnatural base pair systems that are independent of Watson-Crick base pairs and (2) integrating peptide-like functional groups into native nucleobases [12]. However, a significant challenge in using unnatural nucleobases in SELEX is the need for encoding and decoding these nucleotides throughout the selection process, which often requires compatibility with polymerases. Ensuring high accuracy in base pairing selection with or between modified bases during the encoding and decoding stages is another obstacle in nucleobase modifications. To overcome these challenges and expand the range of nucleobase changes available for in vitro aptamer selection, researchers have developed several innovative strategies.

2.2 Ribose modifications

The creation of medicinal antisense oligonucleotides spurred early advances in chemical modifications of aptamers. These changes were made largely to improve resistance to nuclease-mediated degradation [13]. Initially, changes were introduced at the 2′-position of the ribose sugar unit. Modifications at all nucleotides, on the other hand, are seldom tolerated since a sugar alteration reduces aptamer activity [14]. Point-by-point changes and activity testing take time and money. As a result, including changed nucleotides in the SELEX procedure should be investigated. The presence of a polymerase that can be modified is the most significant aspect. Nowadays, replacing fluorine (2′-F) (Figure 2a), methoxy (2′-OMe) (Figure 2b), or amino (2′-NH₂) (Figure 2c) groups for the 2′-hydroxy residue of RNA greatly improves aptamer stability against nuclease degradation [15].

Figure 2.

Chemical structures of modifications for the improvement of nuclease resistance of aptamers: (a) 2′-fluoro-RNA (2′-F); (b) 2′-methoxy-RNA (2′-OMe); (c) 2′-amino-RNA (2′-NH2); (d) Thiophosphorus-DNA/RNA; (e) Phosphorothioate-DNA/RNA.

2′-Aminopyrimidines were utilized in the first ribose-modified SELEX experiment [16]. Then, a change was made to the T7 RNA polymerase to improve substrate compatibility. It has been demonstrated that 2′-fluoro and 2′-deoxypyrimidine are incorporated by T7 RNA polymerase with the Y639F mutation [17, 18, 19]. Pyrimidine alterations have frequently been used as a starting point for aptamer synthesis because they prevent RNase A-mediated degradation [5]. However, in contrast to 2′-fluoropyrimidine, 2′-aminopyrimidine is seldom used in the present SELEX technique due to reduced coupling efficiencies in chemical synthesis, a predilection for the C2′-endo ribose conformation, and a detrimental influence on base pairing stability [20].

Pegaptanib (Macugen®), the sole FDA-approved oligonucleotide-based medicine on the market, exemplifies a sugar-modified aptamer. It was developed in vitro using a combination of unmodified purine nucleotides and 2′-F-pyrimidine nucleotides [9]. The remaining unmodified purine nucleotides were converted to 2′-OMe units through solid-phase synthesis. Additionally, two 2’-F-modified thrombin-binding aptamers (PG13 and PG14) showed a fourfold increase in thrombin-binding affinity and up to a sevenfold improvement in nuclease resistance. In 10% FBS, the improved aptamer’s G-quadruplex stability was enhanced up to 48-fold [21]. Lin et al. created a human neutrophil elastase (HNE) aptamer with a 2’-NH2 group, demonstrating high binding affinity and 10-fold increased stability in human serum and urine compared to the original aptamer [22]. It has also been found that basic fibroblast growth factor (bFGF) can be effectively inhibited by RNA ligands modified with 2′-amino-2′-deoxypyrimidine. When incubated in human serum, 2′-aminopyrimidine-modified RNA aptamers are much more stable than naturally unmodified RNA aptamers (>1000-folds) [23]. In another study by Green et al., Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-binding RNA and DNA aptamers demonstrated a good binding affinity and nuclease resistance [24].

2.3 Phosphate modifications

Modifications to the phosphate portion of aptamer are thought to be a significant technique for aiding aptamer’s resistance to nuclease in vivo. Sulfur can replace two nonbridging oxygens to generate thiophosphorus (PS) (Figure 2d) and phosphorodithioate (PS2) (Figure 2e). Solid-phase synthesis had developed PS-modified DNA. The vulcanization phase replaces the oxidation step in the PS DNA synthesis. Beaucage reagent is used for vulcanizing a recently generated phosphite triester ester. Two isomers would be produced as a result of this reaction [25]. Through high-performance liquid chromatography (HPLC), a single PS modification can be isolated, and synthetic methods can produce several pure diastereomers of PS modifications [26]. The PS modification’s ability to enhance the affinity between aptamers and targets can be illustrated with the following examples.

Wu et al. created two DNA aptamers, XQ-2 and a shortened variant termed XQ-2d, to target Pancreatic Ductal Adenocarcinoma (PDAC), the most frequent pancreatic adenocarcinoma [27]. They made phosphorothioate and 2’-OMe derivatives of XQ-2d to boost serum stability; however, they discovered that the thioaptamer form of XQ-2d had decreased binding to PL45 cells, while the 2’-OMe version did not [28]. Chen et al. investigated a 50-polyethylene glycol (PEG)-modified form of Adipo8 with phosphonothioate linkages placed right after the first base and just before the last base [29]. When examined in tissue culture and in vivo, the addition of these two thioate connections boosted serum stability by a tiny but statistically significant amount. Because this aptamer can identify white adipocytes from brown adipocytes and preadipocytes, it might be used to provide adipocyte-specific treatment [30]. PS2 alterations, which replace both nonbridging oxygen atoms with sulfur, have been incorporated into RNA aptamers chosen for binding to VEGF165. PS2 alterations increase nuclease resistance and aptamer stability in human serum [31].

2.4 Isomerized nucleoside modifications

2.4.1 Spiegelmers

A method for altering aptamers is the creation of “mirror-image” aptamers, also known as spiegelmers (Figure 3a). Spiegelmers counteract nucleases’ stereoselectivity by inverting the chirality centers inside sugar molecules, resulting in a mirror image of wild-type DNA or RNA. This structural change makes spiegelmers more stable in vivo, as they evade recognition by nucleases and the immune system [32]. Traditionally, spiegelmer selection involved a two-step process, where researchers first created a mirror-imaged target and selected D-aptamers using standard SELEX. The chosen D-aptamer was then converted into the corresponding spiegelmer [33]. However, recent advancements in molecular systems capable of replicating and transcribing L-nucleotides have simplified and reduced the cost of the selection process [34].

Figure 3.

Chemical structures of second-generation modifications for the improvement of nuclease resistance of aptamers: (a) Spiegelmers; (b) d−/l-isonucleoside; (c) inverted thymidine; (d) circular Aptamers.

Spiegelmers have found applications in cancer research and therapy. For example, Roccaro et al. developed a spiegelmer targeting SDF-1 to inhibit bone marrow metastasis of multiple myeloma cells [35]. In cell and animal tests, the spiegelmer effectively neutralized SDF-1, demonstrating its potential to inhibit metastasis. NOX-A12, an SDF-1 binding spiegelmer, is currently undergoing phase II clinical trials, showing an 86% response rate against relapsed/refractory chronic lymphocytic leukemia. However, further research is needed before NOX-A12 can be approved as a therapeutically viable drug [33].

2.4.2 d−/l-Isonucleoside modifications

Isonucleosides are nucleoside analogs where the bases are repositioned to the nucleoside’s 2′ or 3′ position (Figure 3b). Oligonucleotides incorporating isonucleosides exhibit increased resistance to nuclease-mediated hydrolysis [36]. Yang et al. explored the potential of d−/l-isonucleosides as modifiers for three aptamers (TBA, GBI-10, and AS1411). The strategic incorporation of d−/l-isonucleosides, particularly in the loop regions, led to notable enhancements in spatial conformation stability and chemical robustness within the modified aptamers [37, 38]. Consequently, the modified aptamers exhibited significantly heightened resistance against biodegradation. Interestingly, modifications with L-isonucleosides had a more profound impact on enhancing the biological activity of the aptamers compared to changes with D-isonucleosides. These findings underscore the significance of isonucleoside chirality in influencing the functional characteristics of modified aptamers.

2.4.3 Inverted nucleoside modifications

Oligodeoxynucleotides were commonly modified with inverted thymidine (5′-,3′-inverted T) (Figure 3c) to render them resistant to nucleases. Pegaptanib also has a 40 kDa poly (ethylene glycol) moiety at the 5′ end to help with renal clearance, as well as a 3′-3′-linked deoxythymidine residue to help with nuclease destruction. Despite these changes, pegaptanib retained an exceedingly high affinity for its VEGF165 target and demonstrated sustained in vitro [39] stability. Moreover, the antifactor IXa RNA aptamer RB06 is composed of unmodified purine nucleosides, 2′-F-pyrimidine nucleosides, 5′-terminal 40 kDa-PEG moiety, and 3′-terminal 3′-inverted deoxythymidine preserved excellent affinity to the target, high in vivo stability and robust anticoagulant efficacy [40].

2.5 Nuclease-resistant circular Aptamers

The creation of circular aptamers (Figure 3d), which solve the difficulty of metabolic instability, is a recent accomplishment in aptamer modification. Aptamers can avoid exonuclease degradation by connecting the 5′ and 3′ termini of nucleic acids to create a closed circular shape, resulting in increased resistance to nucleases [40]. King et al. created multivalent circular aptamers with anticoagulant activity, illustrating the power of cyclization in the creation of functional aptamers [41]. Cyclization enhances aptamer resistance to nucleases, increasing heat stability and guaranteeing structural homogeneity. Tan et al. reported the development of bivalent circular aptamers employing three aptamers that target live cancer cells (Sgc8, TD05, and XQ-2d) [42, 43, 44]. The cyclization technique provides an economical and practical approach to improving the stability and binding ability of aptamers, allowing them to be used in diagnostic and therapy. The usage of circular aptamers is a viable option for increasing aptamer stability and functional qualities, bringing up new possibilities for their use in a variety of biological applications [45].

3. Strategies for prolonging the half-life of aptamers

Due to their quick elimination via renal filtration, tiny aptamers continue to provide a barrier for renal clearance (Figure 4). To solve this, several techniques, including the attachment of cholesterol, PEG, proteins, liposomes, and other materials, have been reported. PEGylation, in particular, is a well-established and commonly utilized technique for prolonging medication half-life. Other approaches, such as lipid nanoparticle delivery systems and attachment to bioactive natural proteins, have also been used to improve the metabolic stability and pharmacokinetic features of therapeutic nucleic acid aptamers. Alternative PEGylation approaches, such as employing long-half-life proteins and low molecular weight coupling agents, have also been investigated to extend the half-life of aptamers. These methodologies may be adapted to specific aptamer applications and therapeutic purposes, allowing for the creation of aptamers with increased stability, bioavailability, and circulation half-life.

Figure 4.

Schematic illustration of chemical modification prevents the aptamer from renal clearance.

3.1 Polyethylene glycol

Polyethylene glycol (PEG) (Figure 5a) is a group of synthetic polymers with high molecular weight, having linear or branched structures and hydroxyl groups [46]. Its molecular weight can vary from a few hundred to tens of thousands, leading to diverse physicochemical characteristics. PEG with a molecular weight below 700 remains liquid at typical temperatures, while PEG exceeding 1000 assumes a predominantly solid form [47]. Thanks to its low toxicity and immunogenicity, the FDA has granted approval for PEG’s pharmaceutical applications. PEGs weighing less than 30 kDa are typically cleared through the kidneys, while those with molecular weights exceeding 20 kDa are excreted in feces [48].

Figure 5.

Chemical structures of modifications for prolonging the half-life of aptamers. (a) Cholesterol-oligonucleotide conjugates; (b) polyethylene glycol (PEG)-oligonucleotide conjugates; (c) dialkylglycerol (DAG)-oligonucleotide conjugates; (d) albumin-oligonucleotide conjugates.

The process of PEGylation involves attaching PEG to macromolecules like proteins, peptides, and nucleic acids. This coating of medication molecules with a hydrophilic shield diminishes immune recognition and enzymatic degradation within the body [49]. Additionally, PEGylation augments the size of drug molecules, thereby reducing renal clearance, as it predominantly relies on the molecule’s size. Consequently, PEGylated drugs often exhibit increased efficacy due to their prolonged biological half-life. Several examples highlight how PEGylation effectively extends the biological half-life of medicinal aptamers. The biological half-life of aptamers is extended differently by different molecular weights of PEG. To reduce renal clearance, the initial version of the von Willebrand factor (VWF) aptamer ARC1779 was modified with a 20 kDa-PEG moiety. However, its limited half-life of approximately 2 hours restricted its clinical application [50]. In contrast, the second-generation VWF aptamer ARC15105 was developed with higher molecular weight PEG moieties (40 kDa), leading to a significantly extended half-life of 66 hours while maintaining the same level of VWF inhibition [51].

Pegaptanib, commercialized as Macugen, is the first FDA-approved PEGylated RNA aptamer for the treatment of age-related macular degeneration. Pegaptanib binds to vascular endothelial growth factor (VEGF) selectively and inhibits its interaction with VEGF receptors, hence reducing neoangiogenesis. Pegaptanib has been modified with a PEG moiety of 40 kDa. Pegaptanib exhibited a half-life of around ten days in clinical studies, and therapy with pegaptanib maintained steady visual acuity in a substantial percentage of patients [52]. NOX-A12, also known as Olaptesed pegol, is an RNA Spiegelmer modified with a 40 kDa branched PEG moiety [53]. It selectively binds to the chemokine CXCL12, hindering its interaction with CXCR4 and CXCR7. This inhibition effectively hampers angiogenesis and metastasis, thereby enhancing cancer treatment. In phase I/II clinical trial, NOX-A12 demonstrated a half-life of 53.2 hours at a dose of 4 mg/kg. Similarly, another PEGylated RNA Spiegelmer, NOX-H94 or lexapeptid pegol, exhibits preferential binding to human hepcidin, leading to the inactivation of its biological function and simultaneously improving blood iron content and transferrin saturation. NOX-H94 was also modified with a 40 kDa branched PEG moiety. It displayed a dose-dependent increase in serum iron content and transferrin saturation, with a half-life ranging from 14.1 to 26.1 hours [54]. These findings illustrate the potential of PEGylated RNA Spiegelmers as promising candidates in various therapeutic applications.

While PEGylation has been frequently used to extend the biological half-life of liposomes for drug administration, repeated injections of PEGylated liposomes can cause an accelerated blood clearance (ABC) phenomenon, shortening the biological half-life. Aside from PEGylation, several methods for prolonging the half-life of nucleic acid aptamers have been investigated, including PAS [55] (proline, alanine, and serine sequences) and PLGA [56] (poly(lactic-co-glycolic acid)). PASylation is accomplished by the use of synthetic polypeptides composed of Pro, Ala, and/or Ser amino acids, which have characteristics similar to PEG. The synthetic polymer PLGA, which is made of lactic acid and glycolic acid, has low toxicity, biodegradability, and biocompatibility. PLGA has been used in nanocarriers for medication delivery systems, including nucleic acid delivery systems. PEGylation has long been the primary approach for increasing medication half-life. However, due to the mixed nature of commercial reagents, their usage has prompted concerns about the consistency of PEGylated medicinal products, as well as safety and bioaccumulation difficulties [57]. Hypersensitivity responses and the development of antibodies against PEG can result in decreased effectiveness in prolonging biological half-life [58]. As a result, various PEGylation techniques have been investigated in order to increase the half-life of biopharmaceuticals. Furthermore, connecting nucleic acid aptamers to bioactive natural proteins is a promising technique with future development potential.

3.2 Albumin

Human serum albumin (HSA) (Figure 5b) is a common protein in human plasma with a high affinity for drug binding (approximately 40 mg/mL). Utilizing its long biological half-life of around 19 days [59] and a molecular weight of 67 kDa, albumin has become increasingly popular as a drug carrier in pharmaceutical applications [60]. Extending the half-life of aptamers can be achieved by improving the blood circulation half-life of HSA through its interaction with the neonatal Fc receptor (FcRn), which facilitates cellular recycling [61]. This approach can enhance the stability and persistence of aptamers in the bloodstream, ultimately leading to improved therapeutic efficacy. Creating albumin-aptamer conjugates through chemical conjugation of aptamers with albumin is a favored method for aptamer administration.

A novel albumin-oligodeoxynucleotide assembly method was developed by Matthias Kuhlmann et al., involving the conjugation of albumin’s cysteine residue at position 34 (cys34) with maleimide-derivatized oligodeoxynucleotides at the 3′ or 5′ end [62]. This construct remained stable in 10% serum over a 24-hour period. Similarly, Julie Schmkel et al. devised a site-specific conjugation technique for attaching an anticoagulant aptamer to recombinant albumins, ensuring the retention of aptamer activity and albumin receptor engagement [63]. Remarkably, the binding affinity of the aptamer conjugate to FcRn was significantly strengthened when it was coupled to recombinant albumin engineered to have enhanced FcRn affinity. These innovative techniques have the potential to significantly alter the pharmacokinetic profile of aptamers, paving the way for improved therapeutic outcomes. Apart from albumin, the human immunoglobulin G (IgG) Fc domain is another long-half-life protein in human blood that can be employed to modify aptamers and enhance their pharmacokinetic characteristics. The IgG Fc domain is commonly utilized to improve the pharmacokinetics of biologically active proteins or peptides [64]. In vivo, IgG exhibits a longer half-life compared to albumin. Therefore, when a drug is conjugated with the Fc domain of IgG, it displays superior pharmacokinetic properties compared to albumin-based conjugates. This modification strategy holds promise for extending the circulation time of aptamers and enhancing their therapeutic efficacy. Furthermore, the Fc domain has a molecular weight equivalent to albumin or PEG. Although no study has been published on using the Fc domain to conjugate aptamers for half-life extension, it is unknown whether Fc domain-modified aptamers have a longer elimination half-life than albumin-modified ones. Aptamer medicines are often exclusively provided through injection. However, because of the high proportion of the macromolecular moiety in the macromolecule-aptamer combination, increasing the subcutaneous dose of the aptamer moiety within a constant subcutaneous administration volume is difficult, limiting its therapeutic potential. As a result, it is critical to overcome the constraint of the dose increase space coming from macromolecular modification techniques.

Another method for modifying aptamers is to use tiny compounds with low molecular weight. Albumin, as previously stated, contains extensive hydrophobic interface cages that may bind particular low molecular weight chemical agents to create molecular complexes. Specific low molecular weight chemical agents can be used to alter aptamers and generate conjugates that bind albumin, resulting in molecular complexes with an average mass greater than the renal filtration cut-off threshold, hence increasing their half-life. Noncovalent binding allows medication attachment to albumin in this technique. Evans Blue (EB), for example, has a strong affinity for albumin and has been demonstrated to have a long half-life in vivo [65]. EB has been found to bind to the hydrophobic region of albumin, leading to the formation of a drug/albumin complex with an extended half-life in circulation and improved physiological stability. Due to this property, EB derivatives have been commonly utilized to enhance the pharmacokinetic properties of peptides and proteins. Researchers have utilized EB to modify Sgc8, an aptamer that specifically targets the PTK7 receptor [66]. Following the conjugation with EB, the aptamer showed a significantly prolonged half-life, and its targeting efficacy was markedly improved when compared to the original unconjugated aptamer. This modification with EB holds great promise for enhancing the therapeutic potential of aptamers and optimizing their in vivo performance. Furthermore, because albumin binds and transports fatty acids, they are often employed for long-term modification. For example, Patients with type 2 diabetes frequently require insulin shots to keep their blood sugar levels steady. However, numerous injections may cause compliance issues, limiting total therapy efficacy. To reduce dose frequency, long-acting glucagon-like peptide-1 receptor agonists (GLP-1RAs) such as liraglutide modified with palmitic acid (half-life of three days) and semaglutide modified with an octadecandioic acid derivative (half-life of seven days) have been created [67]. Jin et al. modified floxuridine homomeric oligonucleotide (LFU20) with a lipid having two fatty acid derivative tails [68]. By utilizing this technique, LFU20 was able to interact with the hydrophobic cavity of albumin, resulting in the formation of an LFU20/albumin complex. This complex exhibited enhanced tumor accumulation, thanks to the improved permeability and retention effect, and was subsequently internalized into the lysosomes of cancer cells. This approach holds great promise for targeted drug delivery and improving the therapeutic efficacy of anticancer agents.

When compared to PEGylation, the fatty acid modification techniques discussed above greatly enhance the fraction of aptamers. Fatty acids, on the other hand, can bind to fatty acid-binding proteins (FABPs), which are found in a variety of tissues throughout the body. This binding may cause drug loss inside those tissues, reducing the quantity of medication in circulation.

3.3 Cholesterol

Cholesterol conjugation (Figure 5c) is an alternate method for improving the pharmacokinetic properties of aptamers. Because low density lipoprotein (LDL) has a strong affinity for cholesterol, cholesterol is an excellent option for alteration. As a result, a cholesteryl-oligonucleotide (cholODN) was created by chemically bonding cholesterol to an aptamer’s 5′ end [69]. Then, in contrast to its unaltered counterpart, the cholODN-LDL complex displayed exceptional resistance to nuclease hydrolysis in serum, resulting in a tenfold extension of half-life.

An RNA aptamer was modified with 2′-F-pyrimidine and connected to cholesterol in one research, resulting in a chol-aptamer [70]. This chol-aptamer was capable of cellular absorption and successfully prevented the replication of Hepatitis C viral RNAs. Importantly, both in vitro and in vivo tests found no evidence of chol-aptamer toxicity. Furthermore, the gene expression profile remained essentially unaltered, notably for typically implicated immune-related genes. Notably, mice who were given the chol-aptamer in vivo showed no obvious problems. The chol-aptamer displayed a considerably longer half-life and a ninefold decrease in plasma clearance rate when compared to unmodified aptamers. The addition of cholesterol to the aptamer improved its hydrophobicity, allowing it to bind to plasma lipoproteins and reduce renal clearance. Preclinical research has shown that cholesterol-conjugated aptamers had a longer circulation half-life and better biodistribution patterns. However, like with PEGylation, cholesterol conjugation may have an effect on the binding affinity of aptamers to their target molecules. As a result, the conjugation location and cholesterol moiety should be optimized.

3.4 Dialkyl lipid (DAG)

Willis et al. developed a way to increase the activity of an aptamer targeting vascular endothelial growth factor (VEGF) by conjugating it with diacylglycerol (DAG) (Figure 5d). The aptamer’s DAG moiety was attached to a lipid tail and integrated into a liposome’s lipid bilayer. This DAG-aptamer-liposome complex performed better. Notably, as compared to the unmodified aptamer, the complex displayed considerably longer plasma retention duration.

4. Conclusions

Aptamers encounter problems such as lower in vivo stability and fast renal excretion. To address these difficulties, researchers used chemical changes to boost aptamer resistance to nucleases and in vivo stability. Modification can be accomplished either during the SELEX procedure (selection process) by adding functional groups to random libraries or after the SELEX methodology has been completed using solid-phase chemical synthesis. The latter approach is especially effective for adding phosphorothioate units that are resistant to nuclease digestion and inhibit fast renal filtration. The limited half-life extension provided by PEG components may require frequent subcutaneous injections, reducing compliance. Developing aptamers with higher proportion of the active aptamer component and employing low molecular weight modification approaches can enhance therapeutic efficacy.

Acknowledgments

This study was supported by Guangdong Basic and Applied Basic Research Foundation (2020A1515110630).

Conflict of interest

The authors declare no conflict of interest.