载siIKKβ脂质纳米粒的制备及其对巨噬细胞再极化的作用

doi:10.6040/j.issn.1673-3770.0.2022.187

摘要/Abstract

摘要： 目的构建负载siRNA-IKKβ的阳离子脂质纳米粒并探索其调节巨噬细胞再极化从而抑制脉络膜新生血管(CNV)的能力。方法使用薄膜分散法制备阳离子脂质纳米粒;通过琼脂糖凝胶电泳筛选最佳基因纳米粒负载比,使用透射电子显微镜和马尔文粒度仪对其进行表征;qRT-PCR检测M2型巨噬细胞中IKKβ及巨噬细胞极化表型相关分子mRNA的表达;Western Blotting检测M2型巨噬细胞内IKKβ蛋白的沉默情况;激光诱导建立小鼠CNV模型并给药;Western blotting检测给药后CNV病灶内巨噬细胞表型的变化;OCTA检测载基因纳米粒对CNV的治疗效果。结果成功制备负载siIKKβ的阳离子脂质纳米粒LNs-siIKKβ,最佳负载比下粒径为(133.3±0.62)nm,Zeta电位为(6.72±0.17)mV;在体外,LNs-siIKKβ抑制M2型巨噬细胞内IKKβ的表达,巨噬细胞产生由M2向M1表型转变的趋势;体内给药后,CNV病灶处巨噬细胞产生由M2向M1表型转变的趋势,且对CNV的生成具有一定的抑制作用;生物安全性良好。结论构建的负载siIKKβ阳离子脂质纳米粒能够在体外及体内CNV病灶处将M2再极化为M1并能抑制CNV的生成。

关键词: 阳离子脂质纳米粒, IκB激酶β, 巨噬细胞极化, 脉络膜新生血管

Abstract: Objective We aim to construct lipid nanoparticles loaded with siRNA targeting IKKβ and explore their ability to inhibit choroidal neovascularization(CNV)by repolarizing the macrophage phenotype. Methods Cationic lipid nanoparticles were prepared by thin film dispersion, and the optimal gene nanoparticle loading ratio was screened for by agarose gel electrophoresis. Particles were characterized using transmission electron microscopy and a Malvern particle sizer; Expression of IKKβmRNA and mRNAs encoding molecules associated with macrophage polarization phenotype in M2 cells was detected via qRT-PCR; Expression of IKKβprotein in M2 cells was detected by Western blotting. A laser-induced CNV model was established in mice and used to investigate treatment. Macrophage phenotypic molecular protein expression in CNV lesions after drug administration was detected by Western blotting. The therapeutic effect of gene loaded nanoparticles on CNV was detected by OCTA. Results LNs-siIKKβ were successfully prepared. Particle size was(133.3±0.62)nm, and zeta potential was(6.72±0.17)mV at the optimal loading ratio. In vitro, macrophages can be repolarized from M2 to M1 phenotype by suppressing IKKβ expression in M2-type macrophages using LNs-siIKKb. In vivo, after treatment, macrophages in CNV lesions tended to repolarize from M2 to M1 phenotype and CNV production was suppressed. Conclusion The constructed siIKKβ-loaded cationic lipid nanoparticles were able to repolarize macrophages from M2 to M1 in vitro and in CNV lesions in vivo, and inhibited the generation of CNV.

Key words: Cationic lipid nanoparticles, IKKβ, Macrophage polarization, Choroidal neovascularization

中图分类号:

R774

初宝睿,曲毅. 载siIKKβ脂质纳米粒的制备及其对巨噬细胞再极化的作用[J]. 山东大学耳鼻喉眼学报, 2023, 37(2): 91-97.

CHU Baorui, QU Yi. Preparation of siIKKβ loaded lipid nanoparticles and assessment of their effect on repolarization of macrophages[J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2023, 37(2): 91-97.

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