Elucidating the uptake and trafficking of nanostructured lipid carriers as delivery systems for miRNA

IF 4.3 3区 医学 Q1 PHARMACOLOGY & PHARMACY European Journal of Pharmaceutical Sciences Pub Date : 2024-11-25 DOI:10.1016/j.ejps.2024.106973
Ivana Ruseska , Amina Tucak-Smajić , Andreas Zimmer
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Abstract

Cationic nanostructured lipid carriers (cNLCs) represent promising non-viral carriers for nucleic acids, such as miRNAs, forming stable self-assembled miRNA complexes due to electrostatic interactions. Prepared by high-pressure homogenization, cNLC formulations, both with and without Nile Red dye demonstrated stable particle sizes in the range of 100–120 nm and positive surface charges (>30 mV), which are necessary for effective cellular uptake. The miRNA complexes formed at mass ratios of 1:2.5 and 1:5 showed similar stability and size, with positive zeta potentials, as well as high cell viability (> 80 %) in 3T3-L1 and MCF-7 cell lines. The cellular uptake studies of miRNA:cNLC complexes in both cell lines revealed that uptake was time- and concentration-dependent, with rapid initial uptake in 30 min and a zig-zag pattern over 24 h. To elucidate the endocytosis mechanism of miRNA:cNLC complexes, 3T3-L1 and MCF-7 cells were incubated with different inhibitors (chlorpromazine, 5-[N-ethyl-N-isopropyl] amiloride, dynasore, nystatin, or sodium azide with 2-deoxy-d-glucose). Results showed significant inhibition of uptake at low temperatures and with ATP depletion, suggesting endocytosis, particularly macropinocytosis, as the main uptake mechanism in 3T3-L1 cells. In MCF-7 cells, the uptake was less inhibited by the substances, indicating the need for more specific methods to fully decipher the endocytic mechanisms involved. Confocal laser scanning microscopy images revealed that the complexes are internalized in vesicles, and are primarily localized in the juxtanuclear region, suggesting trafficking through the endolysosomal system. Colocalization study with LysoTracker™ Green DND-26 showed significant colocalization of miRNA:cNLC complexes with lysosomes in 3T3-L1 cells, indicating trafficking through the endolysosomal system. In MCF-7 cells, colocalization was lower, suggesting macropinocytosis as the primary uptake mechanism. Additional studies showed partial colocalization between labeled NLCs and miRNA, indicating that about 50 % of miRNA is released from NLCs within 30 min post-transfection.

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阐明作为 miRNA 运送系统的纳米结构脂质载体的吸收和迁移。
阳离子纳米结构脂质载体(cNLCs)是很有前途的核酸(如 miRNAs)非病毒载体,可通过静电相互作用形成稳定的自组装 miRNA 复合物。通过高压均质法制备的 cNLC 配方,无论是否含有尼罗河红染料,都显示出稳定的粒度(100-120 nm)和正表面电荷(大于 30 mV),而这正是细胞有效吸收所必需的。以 1:2.5 和 1:5 的质量比形成的 miRNA 复合物显示出相似的稳定性和大小,zeta 电位为正,在 3T3-L1 和 MCF-7 细胞系中的细胞存活率较高(> 80%)。miRNA:cNLC 复合物在这两种细胞系中的细胞摄取研究表明,细胞摄取与时间和浓度有关,30 分钟内开始快速摄取,24 小时内呈 "之 "字形摄取。为了阐明 miRNA:cNLC 复合物的内吞机制,3T3-L1 和 MCF-7 细胞与不同的抑制剂(氯丙嗪、5-[N-乙基-N-异丙基]阿米洛利、达那索、硝司他丁或叠氮化钠加 2-脱氧-D-葡萄糖)进行了孵育。结果表明,在低温和 ATP 耗尽的情况下,摄取会受到明显抑制,这表明内吞作用,尤其是大蛋白内吞作用,是 3T3-L1 细胞的主要摄取机制。在 MCF-7 细胞中,这些物质对摄取的抑制作用较小,这表明需要采用更具体的方法来全面解密其中的内吞机制。共聚焦激光扫描显微镜图像显示,复合物以囊泡形式内化,主要定位于并核区,这表明它们是通过内溶酶体系统运输的。用 LysoTracker™ Green DND-26 进行的共定位研究显示,在 3T3-L1 细胞中,miRNA:cNLC 复合物与溶酶体有明显的共定位,表明它们是通过溶酶体内系统转运的。在 MCF-7 细胞中,共定位程度较低,这表明大蛋白细胞是主要的摄取机制。其他研究显示,标记的 NLC 与 miRNA 之间存在部分共定位,表明转染后 30 分钟内约有 50% 的 miRNA 从 NLC 中释放出来。
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来源期刊
CiteScore
9.60
自引率
2.20%
发文量
248
审稿时长
50 days
期刊介绍: The journal publishes research articles, review articles and scientific commentaries on all aspects of the pharmaceutical sciences with emphasis on conceptual novelty and scientific quality. The Editors welcome articles in this multidisciplinary field, with a focus on topics relevant for drug discovery and development. More specifically, the Journal publishes reports on medicinal chemistry, pharmacology, drug absorption and metabolism, pharmacokinetics and pharmacodynamics, pharmaceutical and biomedical analysis, drug delivery (including gene delivery), drug targeting, pharmaceutical technology, pharmaceutical biotechnology and clinical drug evaluation. The journal will typically not give priority to manuscripts focusing primarily on organic synthesis, natural products, adaptation of analytical approaches, or discussions pertaining to drug policy making. Scientific commentaries and review articles are generally by invitation only or by consent of the Editors. Proceedings of scientific meetings may be published as special issues or supplements to the Journal.
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