## 4 Discussion  
Fluorescence polarization measurements showed that the CMA part of bApt binds to irinotecan, and further, the irinotecan-binding bApt is efficiently incorporated into fibrin gels by binding the TBA part of bApt to thrombin. Furthermore, cell assays revealed that fibrin gels densely containing irinotecan can cause cancer cell death.  
In this study, 29-mer TBA was used at the TBA part of bApt known to have a \(K_{\text{d}}\) value of 0.5 nM for thrombin. This binding to thrombin can delay the blood coagulation reaction by about 16 s compared to the normal condition without any inhibitors [24]. Meanwhile, in the presence of TBAs with different sequences of similar lengths (15-38 mer) with \(K_{\text{d}}\) values of 0.7-126 nM for thrombin, their blood coagulation reactions were delayed by about 2 s [24]. Therefore, the 29-mer TBA sequence used in this study has a relatively high affinity for thrombin and exhibits inhibitory activity although it does not completely inhibit the activity of thrombin.  
Currently, phenomena in which thrombin bound to iron oxide nanoparticles is incorporated into fibrin gels were observed with electron microscopes [31]. Those reports agree with the efficient introduction of bApt into fibrin gels due to the high avidity of the TBA to thrombin and the efficient thrombin-mediated uptake of TBA into fibrin gels observed in this study [26; 30].  
Moreover, in this study, irinotecan was loaded at the CMA site of bApt instead of CPT1. Recently, we have demonstrated that using bApt carrying CPT1, CPT1 was concentrated in a thin layer of about 50 \(\upmu\)m thickness formed by fibrin gels, and thereby the growth inhibitory effect on cancer cells appeared more significantly about 182 times higher than that of CPT1 only [30]. The inhibitory concentration (IC\({}_{50}\)) values of CPT1 entrapped in fibrin gel (7.6 \(\pm\) 0.36 nM) were approximately 170 times lower than that of CPT1 only in the culture medium (1300 \(\pm\) 600 nM) [30]. Similarly, in this study, irinotecan was concentrated in a thin layer formed by fibrin gel and assimilated into the cell, resulting in a higher cancer cell growth inhibitory effect when compared with that obtained only using irinotecan. The IC\({}_{50}\) values of irinotecan for HeLa cell were 1320 \(\pm\) 130 nM [2; 32]. Therefore, the IC\({}_{50}\) values of irinotecan entrapped in fibrin gel were estimated to be approximately several nM to several tens of nM.  
For the application of bifunctional molecular recognition biomolecules to pharmaceuticals, an antibody (emicizumab) with two target recognition sites is currently approved for treating hemophilia [33; 34]. Conversely, for using a nucleic acid aptamer, it has been reported that  
Figure 4: Fluorescence microscopy images of the HeLa cells incubated for 48 h at 37 \({}^{\circ}\)C after addition of (**A**,**E**) fibrinogen and thrombin, (**B**,**F**) fibrinogen, thrombin and CPT1 or irinotecan, (**C**,**G**) fibrinogen, CPT1 or irinotecan, and bApt, and (**D**,**H**) fibrinogen, thrombin, CPT1 or irinotecan, and bApt. The thrombin, CPT1 or irinotecan, and bApt concentrations are 100 nM each and fibrinogen concentration is 1000 nM. Scale bar = 50 \(\upmu\)m.  
aptamer-aptamer conjugates enable signal control of tyrosine kinase receptor [35] or delivery of doxorubicin to the tumor cell/brain lesions [36], and aptamer-antibody or aptamer-aptamer conjugate can attract T and cancer cells and destroy cancer cells [37; 38; 39]; however, they have not been practically used [35; 36; 37; 38; 39].  
The bApt oligonucleotide has the activity of binding to both the anticancer drug and thrombin, and acts as a carrier for anticancer drugs, which enables the concentration of the anticancer drug in fibrin gels around growing cancer cells. In the future, in the design of bApt, the anticancer drug binding site is compatible with other functionalities. Therefore, the concept and methodology may also be applicable to diseases related to blood coagulation in the future.