Liao Xin, Falcon Noelia D, Mohammed Ali A, Paterson Yasmin Z, Mayes Andrew Geoffrey, Guest Deborah J, Saeed Aram
School of Pharmacy, University of East Anglia, Norwich, NR4 7TJ, U.K.
Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU, U.K.
ACS Omega. 2020 Jan 15;5(3):1496-1505. doi: 10.1021/acsomega.9b03216. eCollection 2020 Jan 28.
The natural healing process for tendon repair is associated with high upregulation of collagen type III, leading to scar tissue and tendon adhesions with functionally deficient tendons. Gene delivery systems are widely reported as potential nanotherapeutics to treat diseases, providing a promising approach to modulate collagen type III synthesis. This work investigates a proof-of-concept four-arm cationic polymer-siRNA polyplex to mediate a transient downregulation of collagen type III expression in a tendon cell culture system. The tendon culture system was first supplemented with TGF-β1 to stimulate the upregulation of collagen type III prior to silencing experiments. The four-arm poly[2-(dimethylamino) ethyl acrylate] (PDMAEA) polymer was successfully synthesized via RAFT polymerization and then mixed with siRNA to formulate the PDMAEA-siRNA polyplexes. The formation of the polyplex was optimized for the N:P ratio (10:1) and confirmed by agarose gel electrophoresis. The size and solution behavior of the polyplex were analyzed by dynamic light scattering and zeta potential, showing a hydrodynamic diameter of 155 ± 21 nm and overall positive charge of +30 mV at physiological pH. All the polyplex concentrations used had a minimal effect on the metabolic activity of cultured cells, indicating good biocompatibility. The dose and time effects of the TGF-β1 on collagen type III gene expressions were analyzed by qPCR, showing an optimal dose of 10 ng mL TGF-β1 and 3-fold increase of expression at 48 h in cultured tenocytes. The PDMAEAsiRNA polyplex concept observed a limited yet successful and promising efficiency in silencing collagen type III at 48 h compared to PEI-siRNA. Therefore, this concept is a promising approach to reduce tissue scarring and adhesion following injuries.
肌腱修复的自然愈合过程与III型胶原蛋白的高度上调有关，会导致瘢痕组织以及肌腱粘连，进而使肌腱功能不足。基因递送系统作为治疗疾病的潜在纳米疗法被广泛报道，为调节III型胶原蛋白的合成提供了一种有前景的方法。本研究探讨了一种概念验证型的四臂阳离子聚合物-siRNA多聚体，以介导肌腱细胞培养系统中III型胶原蛋白表达的瞬时下调。在进行沉默实验之前，首先向肌腱培养系统中添加转化生长因子-β1（TGF-β1），以刺激III型胶原蛋白的上调。通过可逆加成-断裂链转移（RAFT）聚合成功合成了四臂聚[2-(二甲氨基)乙基丙烯酸酯]（PDMAEA）聚合物，然后将其与siRNA混合以制备PDMAEA-siRNA多聚体。针对N:P比（10:1）对多聚体的形成进行了优化，并通过琼脂糖凝胶电泳进行了确认。通过动态光散射和zeta电位分析了多聚体的大小和溶液行为，结果表明在生理pH值下，其流体动力学直径为155±21 nm，总正电荷为+30 mV。所有使用的多聚体浓度对培养细胞的代谢活性影响极小，表明具有良好的生物相容性。通过定量聚合酶链反应（qPCR）分析了TGF-β1对III型胶原蛋白基因表达的剂量和时间效应，结果表明在培养的肌腱细胞中，TGF-β1的最佳剂量为10 ng/mL，在48小时时表达增加了3倍。与聚乙烯亚胺（PEI）-siRNA相比，PDMAEA-siRNA多聚体概念在48小时时沉默III型胶原蛋白方面观察到了有限但成功且有前景的效率。因此，这一概念是减少损伤后组织瘢痕形成和粘连的一种有前景的方法。
