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Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Published: February 5th, 2019



1Key Laboratory of Ministry of Education for Developmental Genes & Human Diseases, Institute of Life Sciences and School of Medicine, Southeast University, 2State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials & Devices, School of Biological Science & Medical Engineering, Southeast University
* These authors contributed equally

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Macrophages are a type of innate immune cells distributed in all body tissues, albeit in different amounts. By producing a variety of cytokines and other mediators, they play critical roles in the host defense against invading microbial pathogens, in tissue repair following injury, and in maintaining tissue homeostasis1. Due to their importance, macrophages have continuously been the subject of intensive research. However, despite its prevalence in gene regulation and function studies, siRNA-mediated gene silencing is less likely to succeed in macrophages because these cells—particularly, primary macrophages—are often difficult to t....

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All methods involving live animals were performed in accordance with the animal care and use guidelines of Southeast University, China.

1. Preparation of PEI-SPIONs

  1. Preparation of oleic acid-modified SPIONs
    1. Dissolve FeCl3•6H2O and FeSO4•7H2O in water under the protection of N2.
      1. Add 28 g of FeCl3•6H2O and 20 g of FeSO4•7H2

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The size and zeta potential of PEI-SPIONs prepared with this protocol were in the range of 29 - 48 nm (polydispersity index: 0.12 - 0.23) and 30 - 48 mV, respectively. They were stable in water at 4 °C for over 12 months without obvious aggregation. To evaluate their siRNA binding ability, PEI-SPIONs were mixed with siRNA at various Fe:siRNA weight ratios. Figure 1 shows that when the Fe:siRNA weight ratio reaches 4 and above, the band of free siRNA was .......

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Macrophages are refractory to transfect by commonly used nonviral approaches, such as electroporation, cationic liposomes, and lipid species. Here we described a reliable and efficient method to transfect macrophages with siRNA. Using the present protocol, over 90% of macrophage-like RAW 264.7 cells (Figure 2B) and rat peritoneal macrophages18 can be transfected with siRNA without significant impairment of the cell viability. This method depends on the delivery p.......

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This work was supported by the National Natural Science Foundation of China (81772308) and the National Key Research and Development Program of China (No. 2017YFA0205502).


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Name Company Catalog Number Comments
DMEM Gibco C11995500BT Warm in 37°C water bath before use
Fetal bovine serum Gibco A31608-02
Penicillin/streptomycin (1.5 ml) Gibco 15140122
Tetrazolium-based MTS assay kit Promega G3582 For cytotoxicity analysis
RAW 264.7 cell line Cell Bank of Chinese Academy of Sciences, Shanghai, China TCM13
Tissue culture plates (6-well) Corning 3516
Tissue culture dishes (10 cm) Corning 430167
RNase-free tubes (1.5 ml) AXYGEN MCT-150-C
Centrifuge tubes (15 ml) Corning 430791
Trypsin Gibco 25200-056
Wistar rats Shanghai Experimental Animal Center of Chinese
Academy of Sciences
Bacillus Calmette–Guérin freeze-dried powder National
Institutes for Food and Drug Control, China
for inducing adjuvant arthritis in rats
siRNA GenePharma (Shanghai, China)
Cy3-siRNA RiboBio (Guangzhou, China)
Polyethyleneimine (10 kDa) Aladdin Chemical Reagent Co., Ltd. E107079
Ammonia water Aladdin Chemical Reagent Co., Ltd. A112077
Oleic acid Aladdin Chemical Reagent Co., Ltd. O108484
Dimethylsulfoxide Aladdin Chemical Reagent Co., Ltd. D103272
FeSO4•7H2O Sinopharm Chemical Reagent Co., Ltd 10012118
FeCl3•6H2O Sinopharm Chemical Reagent Co., Ltd 10011918
Dimercaptosuccinic acid Aladdin Chemical Reagent Co., Ltd. D107254
ultrafiltration tube Millipore UFC910096
Tetramethylammonium hydroxide solution Aladdin Chemical Reagent Co., Ltd. T100882
Particle size and zeta potential analyzer Malvern, England Nano ZS90

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