**5. Toxicity**

Toxicity, side effects, and immunogenicity are one of the most important criteria in drug evaluation, apart from shelf life and efficacy of the drug. Aptamers have a significant advantage over antibodies due to their minimal or non-immunogenic nature. Early preclinical studies using Pegaptanib sodium (Macugen), the aptamer drug for preventing wet age related macular degeneration (AMD), did not exhibit any intrinsic toxicity during preclinical studies [37, 41, 42]. The only side effect

**7**

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

induction of cell death and cytokine expression in the same cells [47].

reported arose almost exclusively due to the injection procedure instead of the drug itself [43]. Similarly, other works have also described aptamers as non-toxic thera-

Though aptamers may be inherently non-immunogenic, chemical modification or conjugation may render them immunogenic or cytotoxic. Hence, chemical modification of aptamers works as a double-edged sword. For example, LNAmodified nucleic acids have shown severe hepatotoxicity [46]. 2′-fluoro pyrimidine and 2′-orthomethyl pyrimidine modifications are commonly used in aptamers to introduce endonuclease resistance. 2′-fluoropyrimidine modified RNA aptamers lead to cytotoxicity and induction of interferon-beta expression in human cancer cells *in vitro*. Interestingly, 2′-orthomethyl pyrimidine completely abrogated the

There is a limited toxicological data available on aptamers. Mainly because the field is still in its initial stage and the majority of aptamer-based drugs are still under clinical trials. In comparison, a significant amount of toxicological data is available for antisense oligonucleotides (ASOs). Some cues might be taken from toxicity studies on ASOs; however, we must remember that aptamers are structurally a different class of oligonucleotides; hence, they may not behave in exactly the same way as ASOs. Toxicological properties of ASOs have been described in earlier reviews [48–50]. Less information is available on other class of immunostimulatory oligonucleotides *viz.*, siRNA, gene therapy, and other partially or completely nucleic acid-based molecules. Three major oligonucleotide-based class effects that have been described are: polyanionic effect, the stimulation of innate immunity, and tissue accumulation of oligonucleotide material [51]. These class effects are briefly

Polyanionic effects occur due to the non-specific off-target interaction with proteins that may affect protein function and it usually occurs at a high concentration of ASOs. The toxicological effects of polyanionic effect include activation of the alternative pathway of complement (C′) that results in pseudohypersensitivity responses and inhibition of the coagulation pathway, thus, resulting in delayed clotting. Complement activation has been studied predominantly in non-human primates, and to some extent in humans. Complement activation is a thresholddependent phenomenon and will occur acutely once the threshold is crossed [52]. Stimulation of innate immunity has been reported for ASOs. This activation occurs due to the interaction of Toll-Like Receptor 9 (TLR 9) with the CpG motif within the single-stranded DNA. When CpG motifs are unmethylated, they act as immunostimulants [53]. Unmethylated CpG motifs are common in microbial genomes but occur quite less in vertebrate genome, hence they are called as

pathogen-associated molecular patterns (PAMPs) [54]. CpG PAMP is recognized by pattern recognition receptor TLR9, which is expressed constitutively by B cells and plasmacytoid dendritic cells (pDCs) in humans and other higher primates [55]. Lastly, oligonucleotide accumulation is observed in certain tissues and cells. This accumulated material is termed basophilic granulation, and it is observed most commonly in renal proximal tubular epithelium and tissue macrophages. Accumulation of basophilic granules usually has no effect on associated cells; however, its accumulation at a very high level in cells, especially renal proximal tubular epithelium, may lead to cytotoxicity and eventually organ dysfunction [56].

Apart from the above mentioned three oligonucleotide class effects, there are other effects that should also be studied; however, these three are the most commonly observed and, hence, should be assessed carefully. In a very interesting work, Guo et al. demonstrated that RNA molecules of different shape, size or sequence can initiate immunogenicity at different levels *in vitro* and *in vivo*, both. Findings from this work suggest that properties of RNA nanoparticles may be tuned for safe

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

peutic agents [44, 45].

described below.

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

reported arose almost exclusively due to the injection procedure instead of the drug itself [43]. Similarly, other works have also described aptamers as non-toxic therapeutic agents [44, 45].

Though aptamers may be inherently non-immunogenic, chemical modification or conjugation may render them immunogenic or cytotoxic. Hence, chemical modification of aptamers works as a double-edged sword. For example, LNAmodified nucleic acids have shown severe hepatotoxicity [46]. 2′-fluoro pyrimidine and 2′-orthomethyl pyrimidine modifications are commonly used in aptamers to introduce endonuclease resistance. 2′-fluoropyrimidine modified RNA aptamers lead to cytotoxicity and induction of interferon-beta expression in human cancer cells *in vitro*. Interestingly, 2′-orthomethyl pyrimidine completely abrogated the induction of cell death and cytokine expression in the same cells [47].

There is a limited toxicological data available on aptamers. Mainly because the field is still in its initial stage and the majority of aptamer-based drugs are still under clinical trials. In comparison, a significant amount of toxicological data is available for antisense oligonucleotides (ASOs). Some cues might be taken from toxicity studies on ASOs; however, we must remember that aptamers are structurally a different class of oligonucleotides; hence, they may not behave in exactly the same way as ASOs. Toxicological properties of ASOs have been described in earlier reviews [48–50]. Less information is available on other class of immunostimulatory oligonucleotides *viz.*, siRNA, gene therapy, and other partially or completely nucleic acid-based molecules. Three major oligonucleotide-based class effects that have been described are: polyanionic effect, the stimulation of innate immunity, and tissue accumulation of oligonucleotide material [51]. These class effects are briefly described below.

Polyanionic effects occur due to the non-specific off-target interaction with proteins that may affect protein function and it usually occurs at a high concentration of ASOs. The toxicological effects of polyanionic effect include activation of the alternative pathway of complement (C′) that results in pseudohypersensitivity responses and inhibition of the coagulation pathway, thus, resulting in delayed clotting. Complement activation has been studied predominantly in non-human primates, and to some extent in humans. Complement activation is a thresholddependent phenomenon and will occur acutely once the threshold is crossed [52].

Stimulation of innate immunity has been reported for ASOs. This activation occurs due to the interaction of Toll-Like Receptor 9 (TLR 9) with the CpG motif within the single-stranded DNA. When CpG motifs are unmethylated, they act as immunostimulants [53]. Unmethylated CpG motifs are common in microbial genomes but occur quite less in vertebrate genome, hence they are called as pathogen-associated molecular patterns (PAMPs) [54]. CpG PAMP is recognized by pattern recognition receptor TLR9, which is expressed constitutively by B cells and plasmacytoid dendritic cells (pDCs) in humans and other higher primates [55].

Lastly, oligonucleotide accumulation is observed in certain tissues and cells. This accumulated material is termed basophilic granulation, and it is observed most commonly in renal proximal tubular epithelium and tissue macrophages. Accumulation of basophilic granules usually has no effect on associated cells; however, its accumulation at a very high level in cells, especially renal proximal tubular epithelium, may lead to cytotoxicity and eventually organ dysfunction [56].

Apart from the above mentioned three oligonucleotide class effects, there are other effects that should also be studied; however, these three are the most commonly observed and, hence, should be assessed carefully. In a very interesting work, Guo et al. demonstrated that RNA molecules of different shape, size or sequence can initiate immunogenicity at different levels *in vitro* and *in vivo*, both. Findings from this work suggest that properties of RNA nanoparticles may be tuned for safe

*Role of Novel Drug Delivery Vehicles in Nanobiomedicine*

the threshold of glomerulus cut-off (30–50 kDa) [34].

Renal filtration of small molecule drugs is a phenomenon that needs to be overcome for efficient therapeutic use. Owing to their small size, aptamers also undergo this challenge. An aptamer of 6–30 kDa mass has a size of <5 nm [34]. When an unmodified aptamer is administered intravenously, even using stabilizing backbone modifications, they are subjected to rapid excretion through renal filtration, hence, reduced circulation time. To overcome this challenge, aptamers are functionalized with bulky moieties *viz.*, polyethylene glycol (PEG), liposomes, proteins, cholesterol, organic or inorganic nanomaterials, or multimerized to reach a mass above

PEGylation is one of the most common methods to prevent renal filtration of aptamers. PEG is a flexible, uncharged and a highly hydrophilic polymer that is widely conjugated with therapeutic drugs to reduce reticuloendothelial clearance, extend circulation time and improve drug efficiency [35]. Macugen, which is the only FDA approved aptamer drug in the market is also PEGylated. PEG decreases aggregation and increases the solubility of the conjugates. PEGylation of Macugen increased its half-life to 9.5 –12.5 hours after intravenous and subcutaneous injection, respectively, in the plasma, and up to 94 hours in vitreous humor [36, 37]. Similarly, PEGylation also increased *in vivo* half-life of M7, a DNA aptamer, from <1 to 24–48 hours [38]. This modification did not cause any change in the specificity of M7 aptamer toward its target PD-1 (Programmed death protein 1) and suppressed the growth of PD-L1

(Programmed death-ligand 1) positive colon cancer carcinoma *in vivo* [39].

well tolerated by mice and increased retention time in plasma [14].

A 29 nucleotide-log 2′-F pyrimidine modified RNA aptamer was reported to inhibit Hepatitis C virus (HCV) replication *in vitro*. In the follow-up study, this aptamer was derivatized with cholesterol to form cholesterol aptamer conjugate (chol-aptamer). This conjugate entered the cell and inhibited HCV RNA replication in a cell-based system successfully. Systemic administration of chol-aptamer was

Willis et al. conjugated diacylglycerol (DAG) to the 5′-end of a nuclease resistant VEGF aptamer [40]. This DAG-aptamer conjugate was then incorporated to a liposome bilayer, which resulted in aptamers with improved anti-VEGF activity *in vitro* and *in vivo*, both. Importantly, this DAG-aptamer-liposome complex had a considerably better residence time in plasma as compared to unmodified aptamer [40].

Toxicity, side effects, and immunogenicity are one of the most important criteria in drug evaluation, apart from shelf life and efficacy of the drug. Aptamers have a significant advantage over antibodies due to their minimal or non-immunogenic nature. Early preclinical studies using Pegaptanib sodium (Macugen), the aptamer drug for preventing wet age related macular degeneration (AMD), did not exhibit any intrinsic toxicity during preclinical studies [37, 41, 42]. The only side effect

**4. Renal filtration**

**4.1 PEG attachment**

**4.2 Cholesterol**

**4.3 Dialkyl lipids**

**5. Toxicity**

**6**

use as therapeutic nanocarriers [57]. Findings from this work may be extrapolated to RNA aptamers to some extent.

Conjugated moiety may also cause an adverse affect in aptamer drug. For example, penivacogin, a PEGylated aptamer drug, induced severe allergic reaction during first exposure to the drug during phase III clinical trials. The allergy occurred due to pre-existing anti-PEG antibodies [58]. PEG is widely used in several consumer products including topical and parenteral medications. Possibly, exposure to these wide varieties of products leads to the development of anti-PEG antibodies [35].

Although less clinical data is available to assess the cytotoxicity of aptamers, studies in animal models show low or no cardiotoxicity, hepatotoxicity or renal toxicity. Doxorubicin (DOX), a widely used anticancer drug causes cardiotoxicity as a side-effect due to non-specific uptake. Neither liposomal DOX nor PEGylated DOX has resolved this issue. However, aptamer-DOX conjugate is known to reduce this cardiotoxicity along with increase in efficacy toward tumor suppression in comparison to non-targeted delivery [59] *in vivo*.

The hepatic uptake of aptamer functionalized gold nanostars (Apt-Au NS) was studied by Dam et al. They found high accumulation of the complex in liver; however, there was no hepatic acute toxicity. This was probably due to the high accumulation of the complex in macrophages instead of the hepatocytes [60]. Similarly, other works also demonstrated lack of hepatic and renal toxicity *in vivo* for aptamer-mediated tumor suppression [45, 61].
