**3. Diversity of aptamer library**

*Role of Novel Drug Delivery Vehicles in Nanobiomedicine*

multitudes of vehicles that are being examined.

reduced the clearance by 9-fold [14].

**2.2 Nuclease activity**

We discuss these two below:

tion is a bit challenging.

tance to these molecules [26].

*2.2.2 Post-SELEX*

*2.2.1 In-SELEX*

non-cholesterol-conjugated aptamer, and in accordance, cholesterol conjugation

It is important to note that no one lipid composition induces cytotoxicity in all cell types, i.e., a conjugated lipid may be toxic to one cell type but not to another. Improvements of lipid formulations are constantly explored by the addition of different lipids, targeting molecules, or shielding moieties designed to prevent clearance *in vivo*. Use of neutral lipids for aptamer administration has been explored less due to the difficulty in loading hydrophilic aptamer molecules in the neutral lipids for formulation, as compared to (poly)cationic lipids. Liposomes are just one tool for enhancing the efficacy of aptamers for targeted delivery, and there are

Nuclease activity of the serum is helpful in cleaving foreign DNA, thus, it is a way of prevention from pathogens. However, this activity is deleterious to the oligonucleotide-based therapeutics, including aptamers. Unmodified aptamers may have very short half-lives *in vivo* (<10 minutes) [15]. Two types of nucleases act on these aptamers, *viz.*, exonucleases and endonucleases. Hence, different strategies have been designed to prevent the cleavage activity of each of the nucleases.

In-SELEX and post-SELEX strategies can be utilized to develop nuclease resistant aptamers. Each of these two strategies has its own advantage and limitation.

In this methodology aptamers with the desired modification are generated during the aptamer selection process using modified nucleotides, e.g., modified 2′ sugar position using 2′-amino pyrimidine nucleosides [16, 17], 2′-fluoropyrimidine nucleosides [18, 19], 2′-O-methyl purine [20], and 2′-O-methyl pyrimidine nucleosides [21, 22] and locked nucleic acids (LNAs) [23, 24] have been commonly used for this purpose, preventing resistance from endonucleases. Macugen, the only FDA approved therapeutic aptamer, was also selected using this approach [25]. The Advantage of this methodology is that no further modification is required in the selected aptamers, thus, ruling out the possibility of loss of function due to post-SELEX modification. However, use of unnatural nucleotides posed a challenge to early researchers since wild-type DNA/RNA polymerases could not amplify the unnatural aptamers during preparative PCR round of aptamer selection. However, using synthetic biology researchers developed new polymerases that could amplify oligos with unnatural nucleotides. Still, amplification using these mutant polymerases is not comparable with natural polymerases; hence, aptamer library amplifica-

SOMAmers are another class of aptamers that bear dU residues at the 5′-position. These are uniformly functionalized at the 5′-position with various moieties (e.g., benzyl, 2-naphthyl, or 3-indolyl-carboxamide). These moieties can interact with the target molecule and form novel secondary and tertiary structural motifs within the SOMAmer. Introduction of dU at the 5′-position offers nuclease resis-

Post-SELEX involves the introduction of desired modification in pre-selected aptamers during solid-phase chemical synthesis. This method may often lead

**4**

As we know, aptamer binding to its target depends on its 3-dimensional conformation, which is ultimately governed by its sequence. Hence, an aptamer library with a high degree of sequence variability will have a high variety of structurally variant aptamers; therefore, such a library will have a higher degree of success in developing tight binding aptamers to its target. In contrast to proteins, which are made of 20 different amino acids due to a variety of functional groups, nucleic acids possess a very limited variety of functional groups, i.e., only four bases (adenine, guanine, cytosine and thymine/uracil). Thus, aptamers can be endowed with protein-like properties by addition of functional groups that mimic amino acid side-chains to expand their chemical diversity [26].

SomaLogic, a leading company in aptamer development, pioneered in increasing the repertoire of aptamer sequences in a library. They introduced new functional groups, i.e., hydrophobic side chains (e.g., naphthyl, benzyl, tryptamino and isobutyl) at 5′-position of dUTP nucleotide. Aptamers developed using these modified nucleotides were called Slow Off-rate Modified Aptamers (SOMAmers), and they showed higher success rate in aptamer selection against difficult proteins with success rate going from <30% for unmodified aptamers to >90% for SOMAmers [30].

Kimoto et al. used an extra base pair Ds:Px in the initial library to introduce variety, and developed aptamers with 100-fold higher affinity as compared to the unmodified analogs against VEGF-165 (vascular endothelial growth factor) [31]. Sefah et al. developed introduced non-natural nucleosides Z and P to develop aptamers specifically targeting liver cancer cells [32]. In another work, glycosylated aptamers were used in a method called SELection with Modified Aptamers (SELMA) to develop low-nanomolar affinity aptamers against HIV broadly neutralizing antibody 2G12 [33]. These antibodies protect against HIV by recognizing the unique carbohydrate epitope on HIV. Here, the glycosylated aptamers worked as a scaffold that presented carbohydrate in a manner that mimics their multivalent presentation on HIV, thus it could be used as a vaccine component.
