Fisher Richard K, Mattern-Schain Samuel I, Best Michael D, Kirkpatrick Stacy S, Freeman Michael B, Grandas Oscar H, Mountain Deidra J H
Department of Surgery, University of Tennessee Graduate School of Medicine, Knoxville, Tennessee.
Department of Chemistry, University of Tennessee, Knoxville, Tennessee.
J Surg Res. 2017 Nov;219:136-144. doi: 10.1016/j.jss.2017.05.111. Epub 2017 Jun 27.
We have previously defined mechanisms of intimal hyperplasia that could be targets for molecular therapeutics aimed at vascular pathology. However, biocompatible nanocarriers are needed for effective delivery. Cationic liposomes (CLPs) have been demonstrated as effective nanocarriers in vitro. However, in vivo success has been hampered by cytotoxicity. Recently, neutral PEGylated liposomes (PLPs) have been modified with cell-penetrating peptides (CPPs) to enhance cellular uptake. We aim to establish CPP-modified neutral liposomes as viable molecular nanocarriers in vascular smooth muscle cells.
CLPs, PLPs, and CPP-modified PLPs (R8-PLPs) were assembled with short interfering RNA (siRNA) via ethanol injection. Characterization studies determined liposomal morphology, size, and charge. siRNA encapsulation efficiency was measured via RiboGreen assay. Vascular smooth muscle cells were exposed to equal lipid/siRNA across all groups. Rhodamine-labeled liposomes were used to quantify cell association via fluorometry, live/dead dual stain was used to measure cytotoxicity, and gene silencing was measured by quantitative polymerase chain reaction.
R8-PLPs exhibited increased encapsulation efficiency equivalent to CLPs. PLPs and R8-PLP-5 mol% and R8-PLP-10 mol% had no cytotoxic effect. CLPs demonstrated significant cytotoxicity. R8-PLP-5 mol% and R8-PLP-10 mol% exhibited increased cell association versus PLPs. R8-PLP-10 mol% resulted in significant gene silencing, in a manner dependent on lipid-to-siRNA load capacity.
The negligible cytotoxicity and enhanced cellular association and gene silencing capacity exhibited by R8-PLPs reveal this class of liposomes as a candidate for future applications. Further modifications for optimizing R8-PLPs are still warranted to improve efficacy, and in vivo studies are needed for translational development. However, this could prove to be an optimal nanocarrier for vascular gene therapeutics.
我们之前已经确定了内膜增生的机制，这些机制可能成为针对血管病变的分子治疗靶点。然而，有效的递送需要生物相容性纳米载体。阳离子脂质体（CLP）已被证明在体外是有效的纳米载体。然而，其体内应用因细胞毒性而受到阻碍。最近，中性聚乙二醇化脂质体（PLP）已用细胞穿透肽（CPP）进行修饰，以增强细胞摄取。我们旨在将CPP修饰的中性脂质体确立为血管平滑肌细胞中可行的分子纳米载体。
通过乙醇注入法将CLP、PLP和CPP修饰的PLP（R8-PLP）与小干扰RNA（siRNA）组装在一起。表征研究确定了脂质体的形态、大小和电荷。通过RiboGreen测定法测量siRNA包封效率。所有组均将血管平滑肌细胞暴露于等量的脂质/siRNA中。用罗丹明标记的脂质体通过荧光法量化细胞结合，用活/死双染法测量细胞毒性，并用定量聚合酶链反应测量基因沉默。
R8-PLP表现出与CLP相当的包封效率增加。PLP以及R8-PLP-5 mol%和R8-PLP-10 mol%没有细胞毒性作用。CLP表现出显著的细胞毒性。与PLP相比，R8-PLP-5 mol%和R8-PLP-10 mol%表现出细胞结合增加。R8-PLP-10 mol%导致显著的基因沉默，并依赖于脂质与siRNA的负载能力。
R8-PLP表现出可忽略不计的细胞毒性、增强的细胞结合和基因沉默能力，表明这类脂质体是未来应用的候选者。仍有必要对R8-PLP进行进一步修饰以优化其疗效，并且需要进行体内研究以实现转化发展。然而，这可能被证明是血管基因治疗的最佳纳米载体。
