## 4 Discussion
Furthermore, low-frequency (28 kHz) ultrasound more efficiently releases loaded drugs from nanobots than high-frequency (1 MHz) ultrasound [25], so low-frequency ultrasound should be chosen when fast breakdown of the polymer is required for a high release rate, as in the current study. In addition, sonication-induced polymer breakdown is not permanent; the cross-links in polymers, including alginate, can reform and quickly re-encapsulate the drugs during re-exposure to physiological fluids [20; 21]. Thus, ENB breakdown should be reevaluated to determine whether there is actual binding between EPO and its receptors followed by downstream activation of the neuroprotective cascade from a bioequivalence point of view. Regarding neuronal cell viability following TG treatment, the live/dead fluorescence images and MTS assay results showed that the TG + ENB octreatment with preconditioning sonication and the TG + EPO-octreatment gave similar numbers of live cells and similar cell viabilities (both more than 65.0%) as at 24 h. The neuroprotective actions of EPO have been extensively reported in a variety of in vitro and in vivo brain and spinal cord injury models, including chemical and mechanical models [3; 38; 39; 40; 41]. TG is known to induce ER stress by irreversible inhibition of the sarco-/endoplasmic reticulum Ca\({}^{2+}\)-ATPase, finally leading to apoptosis [27]. Among various chemical neurotoxic agents, such as glutamate, kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) [42; 43; 44], TG is currently the most commonly applied cytotoxic agent. In addition, SH-SY5V cells are the most commonly used human-derived neuronal cell line in neurological studies [26]. Similar to other in vitro EPO experiments in neuronal cell injury models induced by chemical toxins (such as motor neuron injury induced by glutamate and cortical cell injury induced by a histone deacetylase inhibitor [45; 46]), the current experiment revealed that ENBs exerted neuroprotective actions on cell viability similar to those of EPO after sonication-preconditioned autonomous breakdown in the TG-induced in vitro SH-SY5V neuronal cell injury model. To trigger the neuroprotective actions of EPO, the extrahematopoietic EPO receptor signaling pathway should be activated, which subsequently activates downstream mediators including the JAK2/signal transducer and activator of transcription 5 (STAT5) and phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) [5; 37; 47; 48]. In the current study, compared with EPO, ENBs with preconditioning sonication showed delayed and reduced induction of JAK2 expression against TG-induced cytotoxicity before 24 h. This finding might have been caused by the greater time lag for alginate polymer breakdown and subsequent greater EPO release from ENBs than expected [19]. However, at 24 h, nearly the same expression of JAK2 was found, indicating that at least 84.1% breakdown of ENBs is required for full neuroprotective execution. Moreover, in [PERSON] et al. 's study, the time to peak JAK2 expression in the in vitro CNS injury models occurred after approximately 0 to 2 h of treatment [5], but this peak was observed at 6 h in the current study. However, different injury methods were applied (a combination of scratching with a needle tip and kainate treatment versus TG treatment). Either chemical or mechanical agents cause weaker injury modeling than a combination of the two [49]. Furthermore, kainate has 30 times greater neurotoxicity than glutamate, although no neurotoxicity comparison between kainate and TG has been reported [42]. Therefore, TG-induced injury might not have provoked sufficient injury compared with what was expected in the current trial. ER stress-induced apoptosis can be measured with proapoptotic biomolecular markers. TG treatment provokes the accumulation of misfolded proteins, leading to ER stress [50,51].