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Effective delivery of bone morphogenetic protein 2 gene using chitosan-polyethylenimine nanoparticle to promote bone formation

机译：使用壳聚糖 - 聚乙烯亚胺纳米粒子有效地递送骨形态发生蛋白2基因促进骨形成

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摘要

Treating bone defects is still a challenge in clinical practice. Recently, researchers used human bone morphogenetic protein 2 gene (hBMP-2) to induce osteoblast differentiation and promote new bone formation. However, an efficient way to deliver hBMP-2 still needs to be created. In this study, we evaluated whether chitosan-polyethylenimine (CS-PEI) nanoparticle could effectively deliver hBMP-2 locally with lower or no toxicity and promote osteoblast differentiation and new bone formation in vitro and in vivo. Data demonstrated that the synthesized CS-PEI/hBMP-2 nanoparticle at a W/W ratio of 20 to 1, which was the smallest size (162 nm) and highest zeta potential (24 mV), effectively transfected MC3T3-E1 cells without cytotoxicity in vitro, and had the ability to promote cell proliferation. Interestingly, the CS-PEI/hBMP-2 nanoparticle eliminated disadvantages of lower transfection efficiency from chitosan and cytotoxicity from PEI. RT-QPCR data showed that MC3T3-E1 cells treated with CS-PEI/hBMP-2 nanoparticle dramatically expressed higher levels of BMP-2 and significantly increased gene expressions of Col1 on days 3 and 14, Sp7 on days 3, 7, and 14, and ALP on day 14. Alizarin red staining demonstrated that CS-PEI/hBMP-2 nanoparticle-treated MC3T3-E1 cells significantly increased cell mineralization. These in vitro data suggest that the CS-PEI/hBMP-2 nanoparticle can effectively induce osteogenic differentiation of MC3T3-E1 cells in vitro. Western blot analysis further demonstrated that transgene BMP-2 indeed phosphorylated Smad1/5/8, which indicates that CS-PEI/hBMP-2 nanoparticle affects cell differentiation through a BMP-2 signal pathway. Importantly, in vivo data showed that CSPEI/ hBMP-2 nanoparticle clearly promoted new bone formation at the bone defect area 12 weeks post-implantation. This indicates that synthesized CS-PEI/hBMP-2 nanoparticle has the potential to become a useful therapeutic vector for bone defect treatment with further modification.
机译：治疗骨缺损仍然是临床实践中的挑战。最近，研究人员使用人骨形态发生蛋白2基因（HBMP-2）诱导成骨细胞分化并促进新的骨形成。但是，需要创建一种有效的交付HBMP-2的方法。在该研究中，我们评估了壳聚糖 - 聚乙烯亚胺（CS-PEI）纳米粒子是否可以有效地局部地递送HBMP-2，其局部具有较低或无毒性，并在体外和体内促进成骨细胞分化和新的骨形成。数据证明，合成的CS-PEI / HBMP-2纳米颗粒为20-1的比例为20-1，其为最小尺寸（162nm）和最高ζ电位（24mV），有效转染的MC3T3-E1细胞没有细胞毒性体外，并具有促进细胞增殖的能力。有趣的是，CS-PEI / HBMP-2纳米粒子从壳聚糖和PEI的细胞毒性中消除了较低的转染效率的缺点。 RT-QPCR数据显示用CS-PEI / HBMP-2纳米粒子处理的MC3T3-E1细胞显着表达较高水平的BMP-2，并在第3天，7天，SP7和14天，SP7上显着增加了COL1的基因表达和ALP在第14天。茜素红染色证明CS-PEI / HBMP-2纳米颗粒处理的MC3T3-E1细胞显着增加了细胞矿化。这些体外数据表明CS-PEI / HBMP-2纳米颗粒可以有效地诱导体外MC3T3-E1细胞的成骨分化。 Western印迹分析进一步证明了转基因BMP-2的实际磷酸化Smad1 / 5/8，表明CS-PEI / HBMP-2纳米粒子通过BMP-2信号途径影响细胞分化。重要的是，体内数据显示CSPEI / HBMP-2纳米粒子明显促进植入后12周骨缺损区域的新骨形成。这表明合成的CS-PEI / HBMP-2纳米颗粒具有在进一步改性的情况下成为骨缺损治疗的有用治疗载体。

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  • 来源
    《RSC Advances》 |2016年第41期|共9页
  • 作者

    Zhao Liang; Zhang Kai; Bu Wenhuan; Xu Xiaowei; Jin Han; Chang Bei; Wang Banchao; Sun Yingjian; Yang Bai; Zheng Changyu; Sun Hongchen;

  • 作者单位

    Jilin Univ Sch Stomatol Dept Pathol Changchun 130021 Peoples R China;

    Jilin Univ Coll Chem State Key Lab Supramol Struct &

    Mat Changchun 130021 Peoples R China;

    Jilin Univ Sch Stomatol Dept Pathol Changchun 130021 Peoples R China;

    Jilin Univ Sch Stomatol Dept Pathol Changchun 130021 Peoples R China;

    Harbin Med Univ Affiliated Hosp 2 Sino Russian Inst Hard Tissue Dev &

    Regenerat Harbin 150001 Heilongjiang Peoples R China;

    Jilin Univ Sch Stomatol Dept Pathol Changchun 130021 Peoples R China;

    Jilin Univ Sch Stomatol Dept Pathol Changchun 130021 Peoples R China;

    Jilin Univ Affiliated Hosp 2 Changchun 130021 Peoples R China;

    Jilin Univ Coll Chem State Key Lab Supramol Struct &

    Mat Changchun 130021 Peoples R China;

    Natl Inst Dent &

    Craniofacial Res Mol Physiol &

    Therapeut Branch NIH 10 Room 1A01 Bethesda MD 20892 USA;

    Jilin Univ Sch Stomatol Dept Pathol Changchun 130021 Peoples R China;

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