基于介孔硅的姜黄素-siRNA共递送系统构建及其对巨噬细胞M2型极化的影响

doi:10.12016/j.issn.2096-1456.2021.05.003

口腔疾病防治 ›› 2021, Vol. 29 ›› Issue (5): 306-313.DOI: 10.12016/j.issn.2096-1456.2021.05.003

基于介孔硅的姜黄素-siRNA共递送系统构建及其对巨噬细胞M2型极化的影响

李沛汉^1,²(),郎凯^1,²,宋文¹()

1.军事口腔医学国家重点实验室,口腔疾病国家临床医学研究中心,陕西省口腔医学重点实验室,空军军医大学第三附属医院修复科,陕西西安（710032）
2.空军军医大学基础医学院学员二大队七队,陕西西安（710032）

收稿日期:2020-10-28 修回日期:2020-12-24 出版日期:2021-05-20 发布日期:2021-03-08
通讯作者: 宋文
作者简介:李沛汉,本科,Email: lpeihan777@qq.com
基金资助:
国家自然科学基金项目(31800790);陕西省重点研发计划(2019SF-031);陕西省高校科协青年人才托举计划(20190304);陕西省重点科技创新团队(2020TD-033)

Construction of a curcumin-siRNA co-delivery system based on mesoporous silica and its effect on M2-type polarization of macrophages

LI Peihan^1,²(),LANG Kai^1,²,SONG Wen¹()

1. State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Stomatology, Department of Prosthodontics, School of Stomatology, The Air Force Medical University, Xi’an 710032, China
2. The Seventh Team of Second Brigades, College of Basic Medicine, The Air Force Medical University, Xi’an 710032, China

Received:2020-10-28 Revised:2020-12-24 Online:2021-05-20 Published:2021-03-08
Contact: Wen SONG
Supported by:
National Natural Science Foundation of China(31800790);Shaanxi Provincial Key Research and Development Plan Project(2019SF-031);Young Talent fund of University Association for Science and Technology in Shaanxi(20190304);Shaanxi Key Scientific and Technological Innovation Team(2020TD-033)

摘要/Abstract

摘要：

目的构建以介孔硅为基础的姜黄素（curcumin, CUR）-siRNA共递送系统,探讨其对巨噬细胞向M2型极化的促进作用。方法采用常规溶胶-凝胶法制备介孔硅纳米粒（mesoporous silica nanoparticles, MSN）,在其内部孔道吸附小分子CUR,外层吸附阳离子高分子聚乙烯亚胺（polymeric polyethyleneimine, PEI）,与带负电的siRNA结合,形成（CUR@MSN）PEI/siRNA纳米共递送系统;采用透射电镜观察纳米粒制备过程;以不同浓度的纳米粒处理巨噬细胞RAW264.7,采用MTT方法检测细胞活力变化;采用FAM荧光标记的siRNA制备纳米粒后转染细胞,分别采用激光共聚焦扫描显微镜和透射电镜观察纳米粒的细胞摄取;采用靶向GAPDH的siRNA制备纳米粒后转染RAW264.7细胞,观察对靶基因的沉默效率,对照组为未处理细胞组、仅递送CUR组、CUR与siRNA阴性对照（siNC）共递送组,采用实时定量PCR检测沉默效率;进一步采用miRNA-130a-3p反义序列（antisense oligonucleotide, ASO）制备纳米粒转染巨噬细胞RAW264.7,观察对巨噬细胞极化的影响,对照组为未处理细胞组、仅递送CUR组、CUR与miRNA阴性对照（miNC）共递送组,采用Western Blot检测巨噬细胞M2极化标志物精氨酸酶1（arginase 1, Arg-1）的表达。结果（CUR@MSN）PEI/siRNA纳米共递送系统可成功构建;纳米粒呈现剂量依赖性的细胞毒性,在10 μg/mL以下浓度处理组细胞活力在90%以上;纳米粒可被细胞高效摄取,显著抑制靶基因GAPDH的表达,效率约80%（P < 0.05 vs.其余各组）;仅递送CUR或CUR与miNC共递送组细胞Arg-1的表达提升约3倍（P < 0.05 vs.未处理细胞组）,在共递送CUR和ASO后能发挥二者的协同作用,促进巨噬细胞Arg-1表达约8倍（P < 0.05 vs.其余各组）。结论 CUR-siRNA共递送系统可高效转染巨噬细胞实现协同诱导其M2极化的作用。

关键词: 巨噬细胞, M2极化, 精氨酸酶1, 姜黄素, RNA干扰, siRNA/miRNA, 介孔硅纳米粒, 共递送

Abstract:

Objective To fabricate a co-delivery system of curcumin (CUR) and siRNA based on mesoporous silica nanoparticles (MSN) and investigate its potential application in inducing macrophage M2 polarization. Methods MSNs were synthesized using the conventional sol-gel method. The interior mesochannels were occupied by small-molecule CUR, while the exterior surface was adsorbed by cationic polymeric polyethyleneimine (PEI) to link the negatively charged siRNA molecules to formulate the (CUR@MSN)PEI/siRNA co-delivery system. The formulation process was monitored by transmission electron microscopy(TEM). The MTT assay was used to evaluate the cytotoxicity in RAW264.7 cells under various concentrations of nanoparticles. Confocal laser scanning microscopy and TEM were used to observe cell internalization using FAM-labeled siRNA. GAPDH-targeting siRNA was used to prepare nanoparticles and then was transfected into RAW264.7 cells to observe the silencing efficiency of target genes. The knockdown efficiency was examined by real-time quantitative PCR. The related control groups were untreated cells, CUR delivery only and the co-delivery of CUR and siRNA negative control. By loading miRNA-130a-3p antisense oligonucleotide (ASO) to transfect RAW264.7 cells, the effects on the polarization of macrophages were observed. The M2 polarization marker arginase 1 (Arg-1) was measured by western blotting. The related control groups were untreated cells, CUR delivery only and co-delivery of CUR and miRNA negative control. Results The (CUR@MSN)PEI/siRNA co-delivery system was successfully formulated. The nanoparticles exhibited dose-dependent cytotoxicity, and the cell viability was maintained over 90% when the nanoparticle concentration was less than 10 μg/mL. A high cell uptake efficiency was observed, and the target gene knockdown efficiency was greater than 80% (P < 0.05 vs. all the other groups). The CUR delivery-only group and co-delivery of the CUR-and miRNA-negative control group improved Arg-1 expression ~ 3-fold (P < 0.05 vs. untreated cells). Using the co-delivery of CUR and ASO, synergistic effects were obtained, and Arg-1 expression was increased ~ 8-fold (P < 0.05 vs. all the other groups). Conclusion The CUR-siRNA co-delivery system can effectively transfect macrophages and synergistically induce M2 polarization.

Key words: macrophages, M2 polarization, Arg-1, curcumin, RNA interference, siRNA/miRNA, mesoporous silica nanoparticles, co-delivery

中图分类号:

李沛汉,郎凯,宋文. 基于介孔硅的姜黄素-siRNA共递送系统构建及其对巨噬细胞M2型极化的影响[J]. 口腔疾病防治, 2021, 29(5): 306-313.

LI Peihan,LANG Kai,SONG Wen. Construction of a curcumin-siRNA co-delivery system based on mesoporous silica and its effect on M2-type polarization of macrophages[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2021, 29(5): 306-313.

图/表 5

图1 纳米粒制备过程的透射电镜观察及吸附CUR后的释放曲线

Figure 1 Transmission electron microscopy observation during the preparation of nanoparticles and release curve after the adsorption of CUR a: blank mesoporous silica nanoparticles (MSN); b: the curcumin (CUR) adsorption into MSN (CUR@MSN); c: coating of the CUR@MSN surface with polyethylenimine (PEI); d: cumulative CUR release profile from CUR@MSNs in PBS

图2 MTT法检测不同浓度纳米粒处理细胞后细胞活力变化

Figure 2 Cell viability after treatment with various concentrations of nanoparticles by the MTT assay MSN: mesoporous silica nanoparticles; CUR: curcumin; PEI: polyethylenimine

图3 共聚焦显微镜和透射电镜观察细胞摄取（CUR@MSN）PEI/siRNA纳米粒

Figure 3 (CUR@MSN)PEI/siRNA nanoparticle uptake observed by confocal microscopy and TEM a: confocal laser scanning microscopy images of cell uptake (blue-DAPI-labeled nucleus, green-FAM-labeled siRNA, and red-DiI-labeled cell membrane); b: transmission electron microscopy (TEM) images of the transfected cells showed nanoparticle aggregation in endosomes; c: local magnification in the red box shows the aggregation behavior of nanoparticles and degradation of their structures

图4 不同纳米粒转染后巨噬细胞GAPDH基因的表达

Figure 4 GAPDH expression in macrophages after transfection with different nanoparticles *: P < 0.05; MSN: mesoporous silica nanoparticles; CUR: curcumin; PEI: polyethylenimine; siNC: negative control siRNA; siGAPDH: siRNA targeting glyceraldehyde-3-phosphate dehydrogenase

图5 CUR-ASO共递送系统转染巨噬细胞后Arg-1蛋白的表达

Figure 5 Arg-1 protein expression in macrophages after the co-delivery of CUR-ASO a: Western Blot bands; b: relative grayscale of bands calculated by Image J software; MSN: mesoporous silica nanoparticles; CUR: curcumin; PEI: polyethylenimine; miNC: negative control miRNA; ASO: anti-sense oligonucleotides of miR-130a-3p; Arg-1: arginase 1; *: P < 0.05

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基于介孔硅的姜黄素-siRNA共递送系统构建及其对巨噬细胞M2型极化的影响

Construction of a curcumin-siRNA co-delivery system based on mesoporous silica and its effect on M2-type polarization of macrophages

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