Data_Sheet_1_Efficient Delivery of Transducing Polymer Nanoparticles for Gene-Mediated Induction of Osteogenesis for Bone Regeneration.PDF

Developing non-viral gene therapy vectors that both protect and functionally deliver nucleic acid cargoes will be vital if gene augmentation and editing strategies are to be effectively combined with advanced regenerative medicine approaches. Currently such methodologies utilize high concentrations of recombinant growth factors, which result in toxicity and off-target effects. Herein we demonstrate the use of modified cell penetrating peptides (CPPs), termed Glycosaminoglycan (GAG)-binding Enhanced Transduction (GET) peptides with plasmid DNA (pDNA) encapsulated poly (lactic-co-glycolic acid) PLGA nanoparticles (pDNA-encapsulated PLGA NPs). In order to encapsulate the pDNA, it was first condensed with a cationic low molecular weight Poly L-Lysine (PLL) into 30–60 nm NPs followed by encapsulation in PLGA NPs by double emulsion; yielding encapsulation efficiencies (EE) of ∼30%. PLGA NPs complexed with GET peptides show enhanced intracellular delivery (up to sevenfold) and transfection efficiencies (up to five orders of magnitude). Moreover, the pDNA cargo has enhanced protection from nucleases (such as DNase I) promoting their translatability. As an example, we show these NPs efficiently deliver pBMP2 which can promote osteogenic differentiation in vitro. Gene delivery to human Mesenchymal Stromal Cells (hMSCs) inducing their osteogenic programming was confirmed by Alizarin red calcium staining and bone lineage specific gene expression (Q RT-PCR). By combining simplistic and FDA-approved PLGA polymer nanotechnology with the GET delivery system, therapeutic non-viral vectors could have significant impact in future cellular therapy and regenerative medicine applications.

开发兼具保护性与功能性核酸负载递送能力的非病毒基因治疗载体，是将基因增补与基因编辑策略有效整合至先进再生医学疗法中的关键所在。当前此类方法需使用高浓度重组生长因子，这会引发毒性与脱靶效应。本研究证实，将经修饰的细胞穿膜肽（cell penetrating peptides，CPPs，又称糖胺聚糖（Glycosaminoglycan，GAG）结合型增强转导（Glycosaminoglycan-binding Enhanced Transduction，GET）肽）与包封质粒DNA（plasmid DNA，pDNA）的聚乳酸-羟基乙酸共聚物（poly (lactic-co-glycolic acid)，PLGA）纳米粒联合应用的效果。为实现pDNA的包封，首先将其与阳离子低分子量聚-L-赖氨酸（Poly L-Lysine，PLL）缩合形成粒径30~60 nm的纳米粒，再通过双乳化法将其包封于PLGA纳米粒中，最终包封率（encapsulation efficiencies，EE）约为30%。与GET肽复合的PLGA纳米粒可实现显著增强的细胞内递送（最高达7倍）与转染效率（最高达5个数量级）。此外，包封的pDNA负载物可显著提升对核酸酶（如脱氧核糖核酸酶I，DNase I）的抵御能力，从而增强其翻译活性。作为示例，本研究证实此类纳米粒可有效递送质粒骨形态发生蛋白2（pBMP2），该蛋白可在体外促进成骨分化。经茜素红钙染色与骨谱系特异性基因的定量逆转录聚合酶链反应（Q RT-PCR）检测，证实了该载体可将基因递送至人间充质基质细胞（human Mesenchymal Stromal Cells，hMSCs）并诱导其成骨定向分化。通过将简便易用且获美国食品药品监督管理局（Food and Drug Administration，FDA）批准的PLGA聚合物纳米技术与GET递送系统相结合，此类治疗性非病毒载体有望在未来的细胞疗法与再生医学应用中发挥重大作用。