## 4 Discussion  
To examine the feasibility of ENBs for the controlled release of EPO with neuroprotective bioequivalence to EPO as a therapeutic tool against CNS injury, the EPO release ratio after preconditioning sonication was evaluated over 24 h, and the cell viability and biomolecular marker expression were evaluated in vitro following ER stress induced by TG treatment. To manufacture the ENBs (i.e., EPO-MNPs encapsulated by an alginate polymer), 1.0 mL of a 1000 IU rhEPO solution was added to a mixture of 7.5 mg of sodium alginate and 150.0 mg of MNPs, and the mixed solution was sprayed using a nanospray drying technique. The key findings were as follows: (1) one-hour preconditioning sonication at a low frequency caused 61.7% of the EPO to be released from the ENBs at 12 h and 84.1% to be released at 24 h; (2) the SH-SY5V neuronal cell survival ratios were nearly the same in both the TG + EPO and TG + ENB groups over 24 h (more than 65.0% viability at 24 h); (3) EPO receptor signaling pathway activation in the TG + ENB group showed a trend similar to that in the TG + EPO group, but to a lesser degree before 24 h, although these groups had nearly the same result at 24 h; (4) the proapoptotic signaling pathway in the TG + ENB group presented a trend similar to that above, with a slight reduction before 24 h and almost the same level at 24 h compared to that in the TG + EPO group; (5) the apoptotic signaling pathway again showed a similar trend, as the TG + ENB group showed a slightly lower level before 24 h that became very similar to that in the TG + EPO group at 24 h.  
Figure 6: Endoplasmic reticulum stress-induced apoptotic signaling pathway deactivation (**A**) determinded by Western blot analysis and (**B,C**) quantified over 24 h. \(\beta\)-Actin was used as a loading control. TG indicates hapsigargin treatment, TG + EPO indicates thapsigargin and EPO pretreatment, and TG + ENBs indicates thapsigargin and EPO nanobot cotereatment. TGF-\(\beta\), transforming growth factor-beta. * \(p<0.05\), *** \(p<0.001\), and ns, not significant versus TG + EPO cotereatment. One-hour of preconditioning sonication at 50-60 kHz caused a three-fold increase in EPO release from the encapsulating biodegradable alginate polymer compared to the release from the samples without preconditioning sonication (84.1% versus 25.1% at 24 h). This result is similar to the findings in [PERSON] et al. 's report [19], in which 15 min of sonication at 75 kHz increased the rate of \(p\)-nitroaniline release from a biodegradable glycolide polymer by up to 20-fold, and in [PERSON] et al. 's report [33], in which one hour of sonication at 20-70 kHz led to a greater than 10-fold increase in doxorubicin release from a biodegradable diethylacrylamide polymer. Similar to the case for unsoniated drug formulations encapsulated by biodegradable polymers, such as polylactic-co-glycolic acid, the drug release process relies solely on a diffusion mechanism [33; 34]. However, preemptive or on-demand sonication can degrade sodium alginate polymers as well as other biodegradable polymers by disrupting the cross-links, which was expected to increase the release rate of EPO in the current research [19; 20]. Similar to delivery studies with other therapeutics, most EPO delivery trials have focused on the sustained release of loaded EPO over a long period of time in peripheral nervous system injury models to guarantee a stable blood concentration on the concentration-time curve [35; 36]. However, EPO shows a limited therapeutic time window (within 6-8 h) in in vitro models and in vivo small animal models of CNS injury [5; 6; 7; 37]. Therefore, in clinical situations, the delivery and release of EPO should be as fast as possible for effective neuroprotective actions instead of sustained long-term release. This is the reason why fast breakdown of the alginate polymer encapsulating material is important.