**6. Selection process**

Aptamers are selected from a pool of random single-stranded DNA/RNA oligonucleotide library (>1015 random sequences) by iterative rounds of selection and amplification by SELEX method as shown in **Figure 2** [4]. Therapeutic aptamers have been developed against cell surface targets [62] as well as for delivery of therapeutic conjugates into the cells, i.e., for internalization [3]. Though the selection method is fundamentally the same for developing aptamers for either use, the cell-internalizing aptamers require more points to be addressed to ensure a higher degree of success. Endosomal escape and effective delivery to the cytosol is an important aspect that should be considered for the effective biological activity of a cargo. Here we describe current selection methods commonly used for selection of cell-internalizing aptamers, and discuss the limitations of each method. For this purpose, here we compare the two commonly used strategies, i.e., SELEX and cell-SELEX.

#### **6.1 SELEX**

Aptamers have been historically selected against cell surface biomarkers using purified or recombinant proteins. Majority of aptamer therapeutics that are currently in clinical trials have been selected using this methodology. However, there are problems associated with this selection method since the protein against which selection is done might not be in its natural conformation. Proteins are present in physiological milieu in a unique 3D conformation due to either posttranslational modification or their association with other interacting proteins. Proteins that are produced *in vitro* usually don't possess these post-translational modifications, specifically, if they produced in a prokaryotic system, e.g., commonly used *E. coli*. Hence, it is advisable to use eukaryotic expression system *viz.*, mammalian, yeast or insect cells as they would possess post-translational modifications which may

**9**

**Figure 2.**

against the matrix itself.

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

be crucial for the correct 3D conformation for selection of aptamers. However, low protein yield might be an issue if we chose a eukaryotic expression system over the prokaryotic one. There is also a possibility that recombinant cell surface proteins may be insoluble since they need interaction with other cell components (e.g., G-protein coupled receptor), or form multimeric and/or multivalent structures [63, 46]. Lastly, in the majority of cases, aptamer selection is done using only the extracellular domain of the target protein to avoid aptamer selection against the intracellular region of the protein; however, the absence of transmembrane domain may again alter the natural conformation of the protein. Therefore, there is a possibility that aptamers selected using protein-based SELEX approach might not be able to bind their target in a physiological milieu. Liu et al. selected RNA aptamers against histidine-tagged epidermal growth factor receptor variant III (EGFRvIII) ectodomain. This receptor is present in glioblastoma, but not in normal brain tissue. This protein was expressed in an *E. coli* expression system, hence, lacked a specific post-translational modification, i.e., glycosylation. Lack of glycosylation at this particular site resulted in significant alteration of EGFRvIII protein structure and, hence, the selected aptamer was unable to bind this receptor protein in target cells [64]. Using recombinant protein with a tag-normally used for affinity column based purification of a recombinant protein, e.g., His tag-may result in non-specific aptamer selection against the protein tag; hence, it is always recommended to cleave the tag before a purified recombinant protein is used for aptamer selection. Lastly, attachment of a protein to the matrix may result in non-specific aptamer selection

*final selection round. The figure is adapted from Chandola et al. [4].*

*Diagrammatic representation of the systematic evolution of ligands by EXponential enrichment (SELEX) method. A library of ~1015 different single-stranded DNA/RNA molecules is incubated with the target molecules followed by washing. Unbound sequences (non-specific aptamers) are discarded. Bound sequences are recovered and added to the negative control target. Here, the bound sequences (these are aptamers binding to common epitopes) are discarded, and the unbound sequences (target-specific aptamers) are recovered. These sequences are amplified and subjected to further rounds of in vitro selection followed by sequencing after the* 

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

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

#### **Figure 2.**

*Role of Novel Drug Delivery Vehicles in Nanobiomedicine*

comparison to non-targeted delivery [59] *in vivo*.

for aptamer-mediated tumor suppression [45, 61].

to RNA aptamers to some extent.

antibodies [35].

**6. Selection process**

cell-SELEX.

**6.1 SELEX**

use as therapeutic nanocarriers [57]. Findings from this work may be extrapolated

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

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*

Aptamers are selected from a pool of random single-stranded DNA/RNA oligonucleotide library (>1015 random sequences) by iterative rounds of selection and amplification by SELEX method as shown in **Figure 2** [4]. Therapeutic aptamers have been developed against cell surface targets [62] as well as for delivery of therapeutic conjugates into the cells, i.e., for internalization [3]. Though the selection method is fundamentally the same for developing aptamers for either use, the cell-internalizing aptamers require more points to be addressed to ensure a higher degree of success. Endosomal escape and effective delivery to the cytosol is an important aspect that should be considered for the effective biological activity of a cargo. Here we describe current selection methods commonly used for selection of cell-internalizing aptamers, and discuss the limitations of each method. For this purpose, here we compare the two commonly used strategies, i.e., SELEX and

Aptamers have been historically selected against cell surface biomarkers using purified or recombinant proteins. Majority of aptamer therapeutics that are currently in clinical trials have been selected using this methodology. However, there are problems associated with this selection method since the protein against which selection is done might not be in its natural conformation. Proteins are present in physiological milieu in a unique 3D conformation due to either posttranslational modification or their association with other interacting proteins. Proteins that are produced *in vitro* usually don't possess these post-translational modifications, specifically, if they produced in a prokaryotic system, e.g., commonly used *E. coli*. Hence, it is advisable to use eukaryotic expression system *viz.*, mammalian, yeast or insect cells as they would possess post-translational modifications which may

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

**8**

*Diagrammatic representation of the systematic evolution of ligands by EXponential enrichment (SELEX) method. A library of ~1015 different single-stranded DNA/RNA molecules is incubated with the target molecules followed by washing. Unbound sequences (non-specific aptamers) are discarded. Bound sequences are recovered and added to the negative control target. Here, the bound sequences (these are aptamers binding to common epitopes) are discarded, and the unbound sequences (target-specific aptamers) are recovered. These sequences are amplified and subjected to further rounds of in vitro selection followed by sequencing after the final selection round. The figure is adapted from Chandola et al. [4].*

be crucial for the correct 3D conformation for selection of aptamers. However, low protein yield might be an issue if we chose a eukaryotic expression system over the prokaryotic one. There is also a possibility that recombinant cell surface proteins may be insoluble since they need interaction with other cell components (e.g., G-protein coupled receptor), or form multimeric and/or multivalent structures [63, 46]. Lastly, in the majority of cases, aptamer selection is done using only the extracellular domain of the target protein to avoid aptamer selection against the intracellular region of the protein; however, the absence of transmembrane domain may again alter the natural conformation of the protein. Therefore, there is a possibility that aptamers selected using protein-based SELEX approach might not be able to bind their target in a physiological milieu. Liu et al. selected RNA aptamers against histidine-tagged epidermal growth factor receptor variant III (EGFRvIII) ectodomain. This receptor is present in glioblastoma, but not in normal brain tissue. This protein was expressed in an *E. coli* expression system, hence, lacked a specific post-translational modification, i.e., glycosylation. Lack of glycosylation at this particular site resulted in significant alteration of EGFRvIII protein structure and, hence, the selected aptamer was unable to bind this receptor protein in target cells [64]. Using recombinant protein with a tag-normally used for affinity column based purification of a recombinant protein, e.g., His tag-may result in non-specific aptamer selection against the protein tag; hence, it is always recommended to cleave the tag before a purified recombinant protein is used for aptamer selection. Lastly, attachment of a protein to the matrix may result in non-specific aptamer selection against the matrix itself.
