# Multivalent Aptamer Approach: Designs, Strategies, and Applications
## 4 Biosensing and Biomedical Applications
### Biomedical Applications  
Targeted therapeutic-treatment deliveries generally require specific interactions between cell-surface biomarkers and recognition elements [107]. This specific recognition mechanism enables accumulation of therapeutic agents in pathological sites while reducing systemic toxicity [63]. Aptamer-mediated drug-delivery systems take advantage of the enhanced avidities, bio-stability, and intracellular uptake to their targets via multimerization. For instance, [PERSON] et al. [108] used a DNAzyme-driven DNA nanosponge with multivalent aptamers for targeted delivery and controlled release of doxorubicin. In addition, carriers such as aptamer-conjugated DNA self-assemblies can also enable targeted delivery of gene therapies, such as the delivery of siRNA [56; 109], DNAzyme [110], and Cas9/sgRNA (small guide RNA) [111; 112]. It is conceivable that co-deliveries of more than one type of therapeutic agents are also possible.  
Another important area of in vivo multivalent aptamer applications is focused on cancer treatment. Over the past decades, many aptamers that specifically target cancer cells have been successfully selected, including AS1411 (nucleolin-targeted) [113; 114], MUC1 (MUC1-targeted) [115], and Sgc8 (protein tyrosine kinase-7-targeted) [38], among others [39; 74]. However, aptamers have not yet been widely used in in vivo cancer treatments. This is likely, in part, due to their poor performances under physiological conditions. For example, aptamers are known to be vulnerable to nuclease degradations [116]; in addition, conditions such as temperature [61; 117], pH [118], and shear stress [105; 119] have also been shown to affect the abilities of aptamers to bind to their targets. As a promising solution to these challenges, multimerizing aptamers have been used to mitigate in vivo instabilities associated with aptamers, since multimerizing aptamers have been shown to increases steric hindrance, thereby concealing specific cleavage sites from nuclease access [120]. For example, [PERSON] et al. [28] prepared a self-assembled DNA nanocentipede as a multivalent drug carrier by hybridization chain reaction (HCR). This drug carrier remained stable after treatment with Exonuclease III for 2 h, demonstrating significantly increased resistance to degradation.  
In addition, multimerizing aptamers for in vivo applications can enhance the internalization of the delivery vehicle by target cells. Endocytosis is of interest in understanding the mechanisms that guide the entry of nanomaterials into target cells, which is particularly meaningful for in vivo applications [121]. The multivalent effect has been shown to enhance cellular uptake via receptor-mediated endocytosis by improving the local concentration of aptamers [56; 63; 122]. To demonstrate this concept, [PERSON] et al. [123] immobilized multivalent aptamers onto a DNA or a DNA/RNA hybrid structure to build a targeted drug-delivery system. For comparison, the researchers separately conjugated dual-type aptamers (AS1411 and MUC-1) and single-type aptamers onto multiple scaffolds of different levels of valency (i.e., monomers, pentamers, nonamers, or RCA generated multival scaffolds). The RCA-generated multivalent dual-type aptamer system outperformed monomer aptamer systems with a 2.9-fold higher intracellular uptake in serum-free media.