This study developed a three-layer consecutive gene delivery system (T-CGDS) for timely gene delivery into human mesenchymal stem cells (hMSCs). The timing of transcription factor expression is important to effectively induce bone differentiation. Therefore, a three-layered nanocomposite was fabricated using differently sized gold nanoparticles to promote bone regeneration and osteogenic differentiation. The core layer comprised 80 nm gold nanoparticles coupled with ATF4 pDNA. Following coating with heparin-conjugated Pluronic F-127 (HP-F127), 50 nm gold nanoparticles coupled with SP7 pDNA were added to fabricate a bi-layer system. After further coating with HP-F127, 20 nm gold nanoparticles combined with RUNX2 pDNA were added. Consequently, a T-CGDS measuring 350–450 nm was fabricated. Genes were released for more than 8 days, while the size of the T-CGDS decreased over time. When the T-CGDS was applied to hMSCs, the gene in the outer layer (RUNX2) was expressed first, followed by those in the middle (SP7) and core (ATF4) layers. The T-CGDS effectively induced bone differentiation and regeneration in vitro and in vivo. Timely delivery of the ATF4 gene to stem cells via the T-CGDS can greatly assist osteogenic differentiation involved in bone regeneration.

本研究开发了一种三层连续基因传递系统 (T-CGDS)，用于将基因及时传递到人类间充质干细胞(hMSCs) 中。转录因子表达的时机对于有效诱导骨分化很重要。因此，使用不同尺寸的金纳米粒子制造了三层纳米复合材料，以促进骨再生和成骨分化。核心层包含与 ATF4 pDNA 偶联的 80 nm 金纳米颗粒。在用肝素偶联的 Pluronic F-127 (HP-F127) 包被后，加入与 SP7 pDNA 偶联的 50 nm 金纳米颗粒以制造双层系统。用 HP-F127 进一步包覆后，20 nm 金纳米粒子与添加了 RUNX2 pDNA。因此，制造了测量 350-450 nm 的 T-CGDS。基因被释放超过 8 天，而 T-CGDS 的大小随着时间的推移而减少。当 T-CGDS 应用于 hMSCs 时，外层 (RUNX2) 的基因首先表达，然后是中间 (SP7) 和核心 (ATF4) 层的基因。T-CGDS在体外和体内有效诱导骨分化和再生。通过 T-CGDS 将 ATF4 基因及时传递给干细胞可以极大地帮助参与骨再生的成骨分化。