Abstract
Objective
To construct a novel non-viral vector loaded with growth and differentiation factor-5 (GDF-5) plasmid using chitosan, hyaluronic acid, and chondroitin sulfate for osteoarthritis (OA) gene therapy.
Methods
Nano-microspheres (NMPs) were prepared by mixing chitosan, hyaluronic acid, and chondroitin sulfate. GDF-5 plasmid was encapsulated in the NMPs through electrostatic adsorption. The basic characteristics of the NMPs were observed, and then they were co-cultured with chondrocytes to observe their effects on extracellular matrix (ECM) protein expression. Finally, NMPs loaded with GDF-5 were injected into the articular cavities of rabbits to observe their therapeutic effects on OA in vivo.
Results
NMPs exhibited good physicochemical properties and low cytotoxicity. Their average diameter was (0.61±0.20) μm, and encapsulation efficiency was (38.19±0.36)%. According to Cell Counting Kit-8 (CCK-8) assay, relative cell viability was 75%–99% when the total weight of NMPs was less than 560 μg. Transfection efficiency was (62.0±2.1)% in a liposome group, and (60.0±1.8)% in the NMP group. There was no significant difference between the two groups (P>0.05). Immunohistochemical staining results suggested that NMPs can successfully transfect chondrocytes and stimulate ECM protein expression in vitro. Compared with the control groups, the NMP group significantly promoted the expression of chondrocyte ECM in vivo (P<0.05), as shown by analysis of the biochemical composition of chondrocyte ECM. When NMPs were injected into OA model rabbits, the expression of ECM proteins in chondrocytes was significantly promoted and the progression of OA was slowed down.
Conclusions
Based on these data, we think that these NMPs with excellent physicochemical and biological properties could be promising non-viral vectors for OA gene therapy.
摘要
目的
骨关节炎是临床上的一种常见病和多发病。本研究尝试利用壳聚糖、透明质酸和硫酸软骨素为载体,制备一种携载生长分化因子5(GDF-5)质粒的纳米微球用于骨关节炎的基因治疗。
创新点
首次利用壳聚糖、透明质酸、硫酸软骨素三种原料制备可携载GDF-5 质粒的三元纳米微球,并将其应用到骨关节炎的治疗中。
方法
在55 °C 下,按不同比例混合壳聚糖、透明质酸钠、硫酸软骨素和GDF-5 质粒,利用静电吸附原理制备携载GDF-5 质粒的三元纳米微球。分别利用扫描电镜和激光粒度散射仪测试微球的形貌 和粒径;利用凝胶电泳检测质粒与微球的结合情 况;利用CCK-8 检测微球的细胞毒性。将携载 GDF-5 质粒的微球与软骨细胞共培养,并将脂质 体和空载组作为对照组,在预定的时间点通过免 疫荧光染色、免疫组化染色以及生化成分分析, 观察微球对软骨细胞外基质分泌情况的影响。最 后将该纳米微球注射到骨关节炎模型兔体内,通过大体观察、苏木精-伊红(H&E)染色、免疫 荧光染色和免疫组化分析该微球对骨关节炎的 作用。
结论
本研究成功地利用壳聚糖、透明质酸和硫酸软骨素为原料,制备出携载GDF-5 质粒的纳米微球。 其中GDF-5 质粒可以有效地促进软骨细胞外基质的分泌,透明质酸和硫酸软骨素是临床上常见 的治疗骨关节炎的药物。微球具有良好的理化性 能,其细胞毒性小,转染效率高,在体内外均能有效地促进软骨细胞外基质的分泌,能够在一定程度上延缓骨关节炎的进展。该纳米微球将是一种极具希望的可应用于骨关节炎基因治疗的载 体。
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Project supported by the National Natural Science Foundation of China (No. 81201407), the Bureau of Science & Technology and Intellectual Property Nanchong City (Nos. NSMC20170203 and NSMC20170310), the Health and Family Planning Commission of Sichuan Province (No. 17JP496), and the Research Projects of North Sichuan Medical College (No. CBY13-A-ZD09), China
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Chen, Z., Deng, S., Yuan, Dc. et al. Novel nano-microspheres containing chitosan, hyaluronic acid, and chondroitin sulfate deliver growth and differentiation factor-5 plasmid for osteoarthritis gene therapy. J. Zhejiang Univ. Sci. B 19, 910–923 (2018). https://doi.org/10.1631/jzus.B1800095
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DOI: https://doi.org/10.1631/jzus.B1800095
Key words
- Osteoarthritis
- Gene therapy
- Chitosan
- Hyaluronic acid
- Chondroitin sulfate
- Growth and differentiation factor-5 (GDF-5) plasmid