Chen Wenjie, Deng Wei, Xu Xin, Zhao Xiang, Vo Jenny Nhu, Anwer Ayad G, Williams Thomas C, Cui Haixin, Goldys Ewa M
ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Department of Physics and Astronomy, Macquarie University, Sydney, New South Wales 2109, Australia.
J Mater Chem B. 2018 Aug 28;6(32):5269-5281. doi: 10.1039/c8tb00994e. Epub 2018 Aug 1.
Lipid-based nanocarriers with stimuli responsiveness have been utilized as controlled release systems for gene/drug delivery applications. In our work, by taking advantage of the high complexation capability of polycations and the light triggered properties, we designed a novel photoresponsive liposome-polycation-DNA (LPD) platform. This LPD carrier incorporates verteporfin (VP) in lipid bilayers and the complex of polyethylenimine (PEI)/plasmid DNA (pDNA) encoding EGFP (polyplex) in the central cavities of the liposomes. The liposomes were formulated with cationic lipids, PEGylated neutral lipids and cholesterol molecules, which improve their stability and cellular uptake in the serum-containing media. We evaluated the nanocomplex stability by monitoring size changes over six days, and the cellular uptake of the nanocomplex by imaging the intracellular route. We also demonstrated that light triggered the cytoplasmic release of pDNA upon irradiation with a 690 nm LED light source. Furthermore, this light triggered mechanism has been studied at the subcellular level. The activated release is driven by the generation of reactive oxygen species (ROS) from VP after light illumination. These ROS oxidize and destabilize the liposomal and endolysosomal membranes, leading to the release of pDNA into the cytosol and subsequent gene transfer activities. Light-triggered endolysosomal escape of pDNA at different time points was confirmed by a quantitative analysis of colocalization between pDNA and endolysosomes. The increased expression of the reporter EGFP in human colorectal cancer cells was also quantified after light illumination at various time points. The efficiency of this photo-induced gene transfection was demonstrated to be more than double compared to non-irradiated controls. Additionally, we observed a reduced cytotoxicity of the LPDs compared with the polyplexes alone. This study has thus shown that light-triggered and biocompatible LPDs enable an improved control of efficient gene delivery, which will be beneficial for future gene therapies.
具有刺激响应性的脂质基纳米载体已被用作基因/药物递送应用的控释系统。在我们的工作中，利用聚阳离子的高络合能力和光触发特性，我们设计了一种新型的光响应脂质体-聚阳离子- DNA（LPD）平台。这种LPD载体在脂质双层中掺入了维替泊芬（VP），并在脂质体的中心腔中含有聚乙烯亚胺（PEI）/编码增强绿色荧光蛋白（EGFP）的质粒DNA（pDNA）的复合物（多聚体）。脂质体由阳离子脂质、聚乙二醇化中性脂质和胆固醇分子组成，这些成分提高了它们在含血清培养基中的稳定性和细胞摄取能力。我们通过监测六天内的尺寸变化来评估纳米复合物的稳定性，并通过对细胞内途径成像来评估纳米复合物的细胞摄取。我们还证明，在用690nm LED光源照射时，光触发了pDNA的细胞质释放。此外，这种光触发机制已在亚细胞水平上进行了研究。光照后，VP产生活性氧（ROS），驱动了激活释放。这些ROS氧化并破坏脂质体和内溶酶体膜的稳定性，导致pDNA释放到细胞质中并随后进行基因转移活动。通过对pDNA与内溶酶体之间共定位的定量分析，证实了不同时间点pDNA的光触发内溶酶体逃逸。在不同时间点光照后，还对人结肠癌细胞中报告基因EGFP的表达增加进行了定量。与未照射的对照相比，这种光诱导基因转染的效率被证明提高了一倍多。此外，我们观察到与单独的多聚体相比，LPDs的细胞毒性降低。因此，这项研究表明，光触发且生物相容的LPDs能够更好地控制高效基因递送，这将有利于未来的基因治疗。
