**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].

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 secondgeneration VWF aptamer ARC15105 was developed with higher molecular weight

#### **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 Modification Strategies for Enhancing the Metabolic Stabilities and Pharmacokinetics… DOI: http://dx.doi.org/10.5772/intechopen.112756*

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

#### *The Modification Strategies for Enhancing the Metabolic Stabilities and Pharmacokinetics… DOI: http://dx.doi.org/10.5772/intechopen.112756*

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.
