**8. Quality of known aptamers**

Aptamers have generated excitement in the scientific community in the last 2 decades due to their unique characteristics that helped them gain popularity for use in therapeutic purposes. They have been used as both agonists and antagonists targeting cell surface receptors [34]. The Ability of aptamers to specifically bind the cell surface targets catapulted them into the field of targeted delivery of therapeutics. They can be easily conjugated with small molecule drugs, liposomes, proteins, siRNAs, miRNAs, or other nanoparticles. However, when industry leaders in the field of siRNA/miRNA delivery tried to recapitulate the results of siRNA delivery, they failed [81]. Also, since the approval of Macugen, in 2004, by Food and Drug Administration (FDA), no other aptamer-based drug has made to the market. Failure of Fovista (anti-PDGF aptamer to prevent age-related macular degeneration) and Reg1 (an antifactor IXa aptamer paired with its complementary reversal agent, reverseran, to restore blood clotting) failed in phase III clinical trials due to no improvement over current standard care methods has also been disappointing [81]. However, Reg1 also resulted in serious allergies in patients, albeit it was due to conjugated PEG, and not due to the aptamer itself. Though failure of drugs in clinical trials is quite common as many more drugs fail during trials than pass them, but more disappointingly, there is a lack of aptamer-based drugs that are even advancing to the clinic [5]. Unlike antibodies and antibody-based drugs which are supported by big pharmaceutical companies, aptamer development has been largely relegated to academic labs that are often, comparatively, poorly funded.

Levy group observed lack of reproducibility in aptamer binding to its target or therapeutic effects in several cases [81]. This unreliability among novel molecules is a bad precedent that may generate a lack of confidence in an upcoming technology. The poor reproducibility, or robustness, of these aptamers might be due to poor downstream characterizations, which is, in fact, very important for validation of novel aptamers. There is also no standard way of assessing aptamer binding results, as different labs use a variety of methods to assess the binding function of their selected molecules. Hence, there is no straightforward way to compare or validate results between different labs. Other labs often cite the work from previous labs without revalidating the aptamer that they intend to use. Lack of funds, resources or time are often the reason nor not revalidating the aptamers for their own purposes; however, if the aptamer field has to grow, a bit more care and diligence will be needed from the labs that are developing aptamers as well as the end user.

**13**

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

membrane or it might be located in the cytoplasmic region.

dextran sulfate may also be used for the same purpose [5].

Here, we briefly discuss the validations and controls that should be used to ensure a more reliable selection of aptamers for targeted delivery. Soluble proteins or peptides are often used as targets for aptamer selection, and their binding affinity or specificity is confirmed by binding assays. However, further validation using a negative control cell line or a knockout cell line is quite important for such aptamers since aptamers may be selected against an epitope that is buried in the plasma

Varieties of assays are performed to study aptamer binding to target cells, e.g., radioactively or fluorescently labeled aptamers. However, limitation of these assays is that they are performed on bulk cells, hence, it is not known whether the observed signal is due to interaction with the entire cell population, or only a subset of cells. Confocal imaging is often used as a qualitative method to confirm internalization of aptamers, or localization in a particular organelle. Quantification of the signal and statistical methods are often not performed. Since a very small number of cells are typically interrogated, statistical analysis is not performed [81]. The low throughput of imaging, i.e., low cell counting in confocal imaging is usually due to strong lasers that may bleach the fluorescent labeled aptamers. However, with the advent of LED-based high content imaging systems, quantitative analysis in a high-throughput fashion can be easily performed. It is recommended that aptamers should be conjugated to high-quality fluorophores in these studies, i.e., Alexa Fluor dyes or quantum dots instead of traditionally used FITC or TRITC, as these are less resistant to photobleaching. Flow cytometry is also highly recommended to study aptamer internalization due to its high sensitivity, and its ability to interrogate each

It is advisable to limit the incubation time with aptamers during cell-internalization SELEX, typically up to ~1 hour. Incubation with cells for an extended period of time may lead to significant levels of non-specific uptake. It is also recommended to use blocking agents to prevent non-specific binding of aptamers to cell surface. Aptamers are polyanions, therefore, they can readily interact with cell surface moieties due to charge-charge interactions, especially at high concentrations (>1 μM). A Nonspecific polyanionic competitor like ssDNA or tRNA can be used successfully to significantly inhibit non-specific aptamer binding to cells [81]. As an alternative,

Aptamer selection is a repetitive, time-consuming and a laborious process. The immense number of PCR reactions during preparative PCR step for the enrichment of selected aptamers is also a monotonous process which might lead to manual errors. Various tasks in molecular biology such as DNA sequencing, plasmid preparation, and microarray construction have been automated [82, 83]. The automation of these processes has substantially increased the throughput of sample preparation or assays and significantly reduced the amount of time a researcher must spend to

Automation reduces variability during the selection process, leading to better reproducibility of results. Depending on the degree of automation, manpower, time and consumables can be reduced, whereas throughput can be increased by parallelization. The extent of lab automation depends on scope and timeline of the project, and the allocated budget [84]. In principle, there are two concepts of automation, *viz.*, full automation and unit-automation. A full automation pipeline is the one where all steps of the process or assay are performed without any human intervention [85]. In unit automation, human intervention is required in certain

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

cell in a population individually.

**9. Automation of SELEX protocol**

perform purely repetitive mechanical tasks.

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

Here, we briefly discuss the validations and controls that should be used to ensure a more reliable selection of aptamers for targeted delivery. Soluble proteins or peptides are often used as targets for aptamer selection, and their binding affinity or specificity is confirmed by binding assays. However, further validation using a negative control cell line or a knockout cell line is quite important for such aptamers since aptamers may be selected against an epitope that is buried in the plasma membrane or it might be located in the cytoplasmic region.

Varieties of assays are performed to study aptamer binding to target cells, e.g., radioactively or fluorescently labeled aptamers. However, limitation of these assays is that they are performed on bulk cells, hence, it is not known whether the observed signal is due to interaction with the entire cell population, or only a subset of cells. Confocal imaging is often used as a qualitative method to confirm internalization of aptamers, or localization in a particular organelle. Quantification of the signal and statistical methods are often not performed. Since a very small number of cells are typically interrogated, statistical analysis is not performed [81]. The low throughput of imaging, i.e., low cell counting in confocal imaging is usually due to strong lasers that may bleach the fluorescent labeled aptamers. However, with the advent of LED-based high content imaging systems, quantitative analysis in a high-throughput fashion can be easily performed. It is recommended that aptamers should be conjugated to high-quality fluorophores in these studies, i.e., Alexa Fluor dyes or quantum dots instead of traditionally used FITC or TRITC, as these are less resistant to photobleaching. Flow cytometry is also highly recommended to study aptamer internalization due to its high sensitivity, and its ability to interrogate each cell in a population individually.

It is advisable to limit the incubation time with aptamers during cell-internalization SELEX, typically up to ~1 hour. Incubation with cells for an extended period of time may lead to significant levels of non-specific uptake. It is also recommended to use blocking agents to prevent non-specific binding of aptamers to cell surface. Aptamers are polyanions, therefore, they can readily interact with cell surface moieties due to charge-charge interactions, especially at high concentrations (>1 μM). A Nonspecific polyanionic competitor like ssDNA or tRNA can be used successfully to significantly inhibit non-specific aptamer binding to cells [81]. As an alternative, dextran sulfate may also be used for the same purpose [5].
