## 3 Discussion
As aforementioned in this article, NPs are considered to be superior drug delivery vehicles. Specially, amphiphilic block copolymers are naturally inclined to form self-assembled NPs through hydrophobic-lipophilic interactions [25]. PLGA is a widely used biomedical material, but it has weak hydrophilicity and degrades too slowly to meet the needs of drug delivery, and PLGA NPs would be easily removed by the mononuclear phagocytic system (MPS) [36]. By introducing PEG into a PLGA framework, we solved these problems and successfully prepared the desirable PLGA-PEG-PLGA NPs, with efficient DFO encapsulation. Besides, as shown in Figure 3, this NPs vehicle system provided favorable sustained release for DFO. However, a growing body of evidence has revealed that DFO is prone to inactivation. Consequently, to push ahead the application range of this DFO-loaded NPs, the key point is to verify that the encapsulated DFO indeed stays pharmacologically active in a long time course. Hence, in this study, we systematically evaluated the in vitro bioactivities of the controlled released DFO. For hBMSCs, they survived and proliferated well in the DFO-loaded NPs formulation (Figure 4), and the western blot assay indicated that the HIF-1\(\alpha\) expression level was significantly upregulated in the DFO group compared with the blank NPs group or the control group (Figure 5A). By applying qRT-PCR, we observed that the DFO group showed a significantly higher genetic transcription level of VEGF-one of the most important HIF-1\(\alpha\) target genes for angiogenesis-although other monitored genes such as bFGF and TGF-\(\beta\)1 remained unchanged (Figure 5B-D); similarly, the osteogenic genes including osteoclastin and Runx-2 exhibited significant upregulation (Figure 6B,C). Additionally, the ELISA results confirmed a significantly increased VEGF expression, despite not for bFGF or TGF-\(\beta\)1, in protein level (Figure 5E-G); and the ALP activity assessment further revealed that the DFO-loaded NPs continuously enhanced osteogenic differentiation of hBMSCs for the duration of the experiment (Figure 6A). For HUVECs, as shown in Figure 7, DFO-loaded NPs significantly enhanced their angiogenic activity for up to 72 h, which was evidenced by promoted genetic transcription levels of VEGF, ANGPT-2, EGF and PDGF-BB, as well as the bolstered tube formation. Notably, the effect of DFO-loaded NPs on the HUVEC monolayer below resembled the natural paracrine model since HUVECs and the NPs were deliberately segregated using the transwell plate. According to the analyses made above, DFO-loaded NPs have the potential to be an in situ depot for the sustained release of DFO and provide strong angiogenic and osteogenic coupling stimuli for bone regeneration applications. To conclude, this study demonstrated that the PLGA-PEG-PLGA NPs prepared from a modified solvent extraction/evaporation method possessed favorable biocompatibility and could be used as DFO nanocarriers, with a sustained release profile and the in vitro angiogenic and osteogenic properties well preserved in a fairly long time course. However, further in-depth study of the material and in vivo investigations are needed to advance the application of this drug delivery system in the field of bone regeneration.