**9. Automation of SELEX protocol**

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 perform purely repetitive mechanical tasks.

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

*Role of Novel Drug Delivery Vehicles in Nanobiomedicine*

plays a role in it [80].

**8. Quality of known aptamers**

the intracellular receptor-dependent or independent trafficking path, the fate of targeting moiety can be controlled, e.g., macromolecules internalized by clathrindependent RME are mostly destined for lysosomal degradation whereas clathrinindependent internalization leads to endosomal accumulation and sorting to a non-degradative path [74]. However, this method can be used against those targets that have previously been characterized, e.g., nucleolin. Nucleolin is a nuclear ribonucleoprotein that is overexpressed in cancer cells where it shuttles among the cell-surface, cytoplasm and the nucleus. AS1411 aptamer, a G-quadruplex forming oligonucleotide, specifically binds nucleolin and displays antiproliferative activity against cancer cells [79]. However, there are conflicting reports regarding nucleolinmediated uptake of AS1411 since one study suggest that it is nucleolin dependent and the other suggests that it is nucleolin independent. Though the mechanism of escape of AS1411 from endosomes to cytoplasm is unclear, it is likely that its interaction with nucleolin, which shuttles between different cellular compartments,

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.

**12**

stages and only individual stages in a pipeline are partially automated, independent of each other [86]. The advantage of unit-automation is that it keeps a highly open architecture of the pipeline allowing individual modules to be easily modified or exchanged; hence, the pipeline can be easily extended or modified [87].

Unlike the discovery of small molecule-based drugs in industry, where automation is a norm, development of novel aptamers has happened in individual labs in an academic research setting. For truly industrializing the aptamer discovery process, it is imperative to develop high-throughput strategies for aptamer development at different levels, *viz.*, aptamer selection process, sequencing, and characterization of aptamers involving affinity and specificity studies toward its target. Here we discuss about the high-throughput technologies which have been developed facilitating high-throughput selection of aptamers.

The first attempt to automate aptamer selection process was performed by the group of Ellington [82] followed by some other improvements [88–92]. It brought down the selection time from weeks or months to days. Automation of the SELEX process increased the throughput of selections, but shifted the bottleneck within a selection pipeline toward the identification and evaluation side.

In 2011 the group of T.M. Soh developed a Quantitative Parallel Aptamer Selection System (QPASS), which integrates microfluidic aptamer selection (M-SELEX) and next-generation sequencing (NGS) with in situ synthesized aptamer arrays, enabling simultaneous measurement of affinity and specificity of several 1000 candidate aptamers in parallel. Major advantage of this high-throughput platform is that it automated the characterization, i.e., finding the affinity and specificity of aptamers which is a major bottleneck in aptamer selection, since traditionally the affinity and specificity of aptamers is measured individually in a serial manner [93]. This group used angiopoietin-2 (Ang2) as a target and performed four rounds of M-SELEX followed by next-generation sequencing to obtain enriched aptamer sequences. Next, using aptamer arrays they simultaneously measured dissociation constant (*Kd*) of ∼1000 aptamer candidates in parallel, identifying six high-affinity Ang2 aptamers with Kd <30 nM.

Development of NGS platform in the last decade has revolutionized the aptamer selection process. The Schroeder group in 2010 made the initial attempt in this direction as they combined genomic SELEX with high-throughput sequencing for the identification of genomic aptamers [94]. The same year, Soh group (2010) integrated microfluidic SELEX with high-throughput sequencing to obtain novel aptamers against platelet-derived growth factor BB (PDGF-BB) protein in just three rounds of selection, which traditionally takes 12–20 rounds [95]. Advantage of HTS is that it can track the copy number and enrichment fold of more than 10 million individual aptamer sequences through multiple rounds of selection. This enables the identification of high-affinity aptamers without the need of fully converging the aptamer pool to a small number of sequences, which is required for cloningbased selection method. Similarly, Hoon et al. obtained high-affinity aptamers after just one round of positive selection followed by HTS [96].
