Puras G, Martínez-Navarrete G, Mashal M, Zárate J, Agirre M, Ojeda E, Grijalvo S, Eritja R, Diaz-Tahoces A, Avilés-Trigueros M, Fernández E, Pedraz J L
Neuroprothesis and Neuroengineering Research Group, Miguel Hernández University , 03202 Alicante, Spain.
Institute for Advanced Chemistry of Catalonia, IQAC-CSIC, Spanish Council for Scientific Research , 08034 Barcelona, Spain.
Mol Pharm. 2015 Oct 5;12(10):3658-71. doi: 10.1021/acs.molpharmaceut.5b00422. Epub 2015 Sep 11.
The present study aimed to evaluate the incorporation of protamine into niosome/DNA vectors to analyze the potential application of this novel ternary formulation to deliver the pCMS-EGFP plasmid into the rat retina. Binary vectors based on niosome/DNA and ternary vectors based on protamine/DNA/niosomes were prepared and physicochemically characterized. In vitro experiments were performed in ARPE-19 cells. At 1:1:5 protamine/DNA/niosome mass ratio, the resulted ternary vectors had 150 nm size, positive charge, spherical morphology, and condensed, released, and protected the DNA against enzymatic digestion. The presence of protamine in the ternary vectors improved transfection efficiency, cell viability, and DNA condensation. After ocular administration, the EGFP expression was detected in different cell layers of the retina depending on the administration route without any sign of toxicity associated with the formulations. While subretinal administration transfected mainly photoreceptors and retinal pigment epithelial cells at the site of injection, intravitreal administration produced a more uniform distribution of the protein expression through the inner layers of the retina. The protein expression in the retina persisted for at least one month after both administrations. Our study highlights the flattering properties of protamine/DNA/niosome ternary vectors for efficient and safe gene delivery to the rat retina.
本研究旨在评估将鱼精蛋白掺入非离子型脂质体/DNA载体中，以分析这种新型三元制剂在将pCMS-EGFP质粒递送至大鼠视网膜方面的潜在应用。制备了基于非离子型脂质体/DNA的二元载体和基于鱼精蛋白/DNA/非离子型脂质体的三元载体，并对其进行了物理化学表征。在ARPE-19细胞中进行了体外实验。在鱼精蛋白/DNA/非离子型脂质体质量比为1:1:5时，所得三元载体尺寸为150 nm，带正电荷，呈球形形态，可凝聚、释放并保护DNA免受酶消化。三元载体中鱼精蛋白的存在提高了转染效率、细胞活力和DNA凝聚。眼内给药后，根据给药途径在视网膜的不同细胞层中检测到EGFP表达，且没有与制剂相关的任何毒性迹象。视网膜下给药主要在注射部位转染光感受器和视网膜色素上皮细胞，而玻璃体内给药则使蛋白质表达在视网膜内层更均匀地分布。两种给药后视网膜中的蛋白质表达持续至少一个月。我们的研究突出了鱼精蛋白/DNA/非离子型脂质体三元载体在高效、安全地将基因递送至大鼠视网膜方面的良好特性。
