**2. Serum stability**

*Role of Novel Drug Delivery Vehicles in Nanobiomedicine*

modification that allows conjugation with a variety of molecules and stability at a higher temperature. Aptamer is comparably smaller (~3 nm) than an antibody (10–15 nm) [3], thus, allowing higher penetration in tissues and the ability to bind more dense cellular epitopes in living cells. Unlike antibodies, aptamers can be selected against non-immunogenic molecules, including proteins or peptides as well as toxins. Aptamers have been successfully developed against proteins, peptides, dyes, metal ions, viruses, bacteria, toxins, and whole cells. They have high specificity and affinity often comparable with antibodies. Unlike antibodies, they can be selected at non-physiological conditions such as extremely high or low temperature, or pH. Synthetic manufacturing of aptamers allows minimal batch-to-batch variation, which is a tedious task to maintain in antibody development. All these properties make aptamers as suitable candidates for therapeutic use, particularly targeted nano-delivery. Several aptamers have already been selected against cell surface targets for targeted delivery of therapeutic payload for different diseases, e.g., cancer and human immunodeficiency virus (HIV) infected cells [4]. **Table 1**

lists the common differences between antibodies and aptamers.

Size 10–15 nm ~3 nm

Specificity High High Affinity High High

be raised against toxic or nonimmunogenic proteins

repeated freeze-thaw causes

Synthesis Only in physiological conditions Synthetic

facility or reactors

Scale-of-synthesis Low Scalable

*This table lists the common differences between an antibody (IgG) and an aptamer.*

Cost of synthesis High. Requires animal house

Target Only immunogenic targets. Can't

Penetration Low tissue penetration due to large size

Shelf life Few months (at low temperature;

loss-of function)

Stability (pH, Temperature)

Nuclease susceptibility

Batch-to-batch variation

In this book chapter, we discuss the challenges that hinder the clinical use of aptamers as targeted delivery molecules. For example, the small size of aptamers is like a double-edged sword. Though it helps in higher tissue penetration, but also results in high renal filtration that causes loss of its therapeutic efficiency. Using examples we show that how challenges in serum stability, renal filtration, selection

**Antibody (IgG) Aptamer**

Low High

Immune response High (except humanized Ab) Absent or low (rare cases)

Conjugation Less possibilities Easy conjugation due to chemical nature

Produced against immunogenic and nonimmunogenic (e.g., metal ions, dyes, small

High tissue penetration due to small size

Several months (at room temperature) to

allows functionalization with a wide variety

Low. Synthesized using table-top

peptides, toxins etc.) targets

several years (frozen)

nuclease susceptibility)

of molecules

instruments

Absent Present (modified nucleotides minimize

Present Absent due to synthetic manufacture

**2**

**Table 1.**

Currently, many aptamers are in different stages of clinical trials for various diseases [5]. Most of the drugs are administered systemically, i.e., intravenously. This method poses a challenge of overcoming serum stability, which involves overcoming nuclease activity and coagulation. Here in we discuss these two topics in detail:

#### **2.1 Aggregation**

Aptamers have been used to functionalize liposomes to increase their bioavailability through targeted delivery (**Figure 1**) [4]. Since aptamers are negatively charged it is easier to pack them with cationic liposomes. However, serum proteins being charged molecules bind to cationic lipids, thus, lowering their targeting efficiency due to change in structure, aggregation or dissociation of the aptamercationic lipid complex [6, 7–9]. Polycationic lipids have also shown cyto(toxic) effects [10]. The problem of aggregation incurred by the charge of cationic lipids can be overcome by using neutral helper lipids [11, 12]. But another challenge is that lipid conjugated aptamers may also bind to erythrocytes, and filtered out through liver and spleen. For example, 1,2-dioleoyl phosphatidylethanolamine (DOPE), a neutral helper lipid, binds to erythrocytes. As an alternative, another neutral helper lipid, cholesterol, may be used which shows significantly lower binding to erythrocytes as compared to DOPE [10, 13].

Cholesterol conjugated RNA aptamer against Hepatitis C virus (HCV) NS5B protein has been used to inhibit HCV replication in human liver cells without induction of cytotoxicity *in vitro*. Next, in this study, the same molecule was injected in a mouse model but found no induction of innate immunity. The half-life of the cholesterol-conjugated aptamer was longer than that of the

#### **Figure 1.**

*Illustration showing (a) non-functionalized liposome and (b) aptamer-functionalized liposome carrying drug molecules.*

non-cholesterol-conjugated aptamer, and in accordance, cholesterol conjugation reduced the clearance by 9-fold [14].

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 multitudes of vehicles that are being examined.

#### **2.2 Nuclease activity**

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. We discuss these two below:

#### *2.2.1 In-SELEX*

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 amplification is a bit challenging.

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 resistance to these molecules [26].

#### *2.2.2 Post-SELEX*

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

**5**

*Aptamers for Targeted Delivery: Current Challenges and Future Opportunities*

to reduced affinity or specificity of the aptamers due to the structural change in the aptamer that is brought upon by the modification. Hence, a combination of methods has to be tested on the aptamer, and one that renders no loss of function has to be selected. Modification of 5′ and 3′ nucleotides is most common and effective in this case. Sometimes unmodified aptamers may demonstrate high resistance to serum nuclease activity [27]. This feature might develop due to the unique 3-D conformation of the aptamer that protects the 5′- and 3′-termini from

Spiegelmers are excellent nuclease resistant *L*-form RNA or DNA aptamers that are chiral inversions of the natural D-forms. Since naturally occurring nucleases can act only on *D*-form of nucleotides, they are very less likely to degrade the *L*-form spiegelmers [28]. Also, due to their unnatural conformation, they are less likely to generate an immune response. The unnatural conformation of spiegelmers also renders it unfit for processing by polymerases for amplification during SELEX process. Hence, in the first step, an aptamer library of natural D-oligonucleotides is selected against a synthetic enantiomer of the *L*-protein target. After the aptamer selection is done, aptamers are converted from *D*-form to *L*-form. As per the rules of symmetry, the resulting *L*-aptamers (spiegelmers) would bind to the natural *D*-protein target with the same affinity as the *D*-aptamers bind to the mirror image

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

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

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.

*DOI: http://dx.doi.org/10.5772/intechopen.84217*

exonucleases.

selection target [29].

SOMAmers [30].

**3. Diversity of aptamer library**

side-chains to expand their chemical diversity [26].

#### *Aptamers for Targeted Delivery: Current Challenges and Future Opportunities DOI: http://dx.doi.org/10.5772/intechopen.84217*

to reduced affinity or specificity of the aptamers due to the structural change in the aptamer that is brought upon by the modification. Hence, a combination of methods has to be tested on the aptamer, and one that renders no loss of function has to be selected. Modification of 5′ and 3′ nucleotides is most common and effective in this case. Sometimes unmodified aptamers may demonstrate high resistance to serum nuclease activity [27]. This feature might develop due to the unique 3-D conformation of the aptamer that protects the 5′- and 3′-termini from exonucleases.

Spiegelmers are excellent nuclease resistant *L*-form RNA or DNA aptamers that are chiral inversions of the natural D-forms. Since naturally occurring nucleases can act only on *D*-form of nucleotides, they are very less likely to degrade the *L*-form spiegelmers [28]. Also, due to their unnatural conformation, they are less likely to generate an immune response. The unnatural conformation of spiegelmers also renders it unfit for processing by polymerases for amplification during SELEX process. Hence, in the first step, an aptamer library of natural D-oligonucleotides is selected against a synthetic enantiomer of the *L*-protein target. After the aptamer selection is done, aptamers are converted from *D*-form to *L*-form. As per the rules of symmetry, the resulting *L*-aptamers (spiegelmers) would bind to the natural *D*-protein target with the same affinity as the *D*-aptamers bind to the mirror image selection target [29].
