用于向结肠靶向siRNA递送的生姜脂质纳米粒的制备与表征

Junsik Sung; Chunhua Yang; James F. Collins; Didier Merlin

doi:10.21769/BioProtoc.3685

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Preparation and Characterization of Ginger Lipid-derived Nanoparticles for Colon-targeted siRNA Delivery

用于向结肠靶向siRNA递送的生姜脂质纳米粒的制备与表征

JS Junsik Sung email

CY Chunhua Yang

JC James F. Collins

DM Didier Merlin

发布: 2020年07月20日第10卷第14期 DOI: 10.21769/BioProtoc.3685 浏览次数: 3504

评审: David PaulGANG YUAnonymous reviewer(s)

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Abstract

Synthetic nanoparticle-based drug delivery system is widely known for its ability to increase the efficacy and specificity of loaded drugs, but it often suffers from relatively higher immunotoxicity and higher costs as compared to traditional drug formulations. Contrarily, plant-derived nanoparticles appear to be free from these limitations of synthetic nanoparticles; they are naturally occurring biocompatible vesicles that do not generate immunotoxicity and are easy to obtain. Additionally, lipids isolated from plant-derived nanoparticles have shown the capability of assembling themselves to spherical nano-sized liposomal particles. Herein, we employ lipids extracted from ginger-derived nanoparticles and load them with therapeutic siRNA (CD98-siRNA) to create CD98-siRNA/ginger-lipid nanoparticles. Characterization of the CD98-siRNA/ginger-lipid nanoparticles showed that they present a spherical shape, with a diameter of around 189.5 nm. The surface zeta potential of the nanoparticles varies from -18.1 to -18.4 mV. Furthermore, in recent research, the CD98-siRNA/ginger-lipid nanoparticles have shown specific colon targeting capability and excellent anti-inflammatory efficacy in a Dextran Sodium Sulfate (DSS) induced mouse model of colitis.

Keywords: Ginger-derived nanoparticles (姜源纳米颗粒)

Thin-film hydration (薄膜水合作用)

siRNA delivery (siRNA 递送)

Anti-inflammatory (消炎)

Colon-targeted (结肠靶向)

Background

Small interference RNAs (siRNAs) are a type of promising therapeutic agents that can treat various diseases by silencing the abnormally upregulated messenger RNAs (mRNAs) (Nikam and Gore, 2018). Despite the effectiveness of siRNAs, the safe and efficient delivery of the siRNAs to the therapeutic targets is still a challenging task (Tatiparti et al., 2017). Studies have shown that artificially synthesized nanoparticles can be used to target low doses of siRNAs to specific colonic cell types (e.g., epithelial cells and macrophages) (Zhang et al., 2016b). However, artificially synthetic nanoparticles have several limitations, such as potential in vivo immune toxicity and formidable production cost. What is currently needed is a safer and more economical platform for siRNA-based drug delivery.

Our laboratory and others have recently shown that plant-derived nanoparticles are capable of specifically target the colon, and these naturally occurring nanoparticles are safer and cheaper than synthetic nanoparticles (Zhang et al., 2016a). Additionally, we can extract lipids from these plant-derived nanoparticles and reassemble them to siRNA-incorporated nanoparticles, creating a colon-targeting siRNA/lipid nanoparticle (Zhang et al., 2017). In the following protocol, we will use ginger-derived nanoparticles and CD98-siRNA as starting materials to demonstrate the process of making CD98 siRNA nanoparticles (CD98-siRNA/ginger-lipid). The nanoparticles prepared in this protocol have shown its effectiveness in treating ulcerative colitis in recent research (Zhang et al., 2017).

Materials and Reagents

Pipette tips:
0.1-10 μl (Sorenson Bioscience, catalog number: 70600 )
1-200 μl (Sorenson Bioscience, catalog number: 70520 )
100-1,000 μl (Sorenson Bioscience, catalog number: 70540 )
1.5 ml Eppendorf Safe-Lock tubes (Eppendorf, catalog number: 0 22363204 )
22 ml culture tubes, borosilicate glass (VWR, catalog number: 47729-580 )
15 ml conical tubes (Denville Scientific, catalog number: C1018-P )
50 ml conical tubes (Denville Scientific, catalog number: C1062-P )
Polycarbonate centrifuge tubes (Beckman Coulter, catalog number: 349622 )
4.5 ml polystyrene cuvettes, square (Brookhaven Instruments Corps., catalog number: BI-SCP )
200 nm polycarbonate membrane (Millipore, catalog number: GTTP01300 )
Formvar^®-coated copper grids (Electron Microscopy Sciences, catalog number: FCF300-CU-SC )
Filter paper (VWR, catalog number: 28313-068 )
Mica sheet (Electron Microscopy Sciences, catalog number: 71855-15 )
Kimwipes (Kimberly-Clark, catalog number: 06-666 )
Capillary cell and plastic cap (Malvern, catalog number: DTS 1070 )
Mouse (Jackson Laboratory, model: FVB/NJ 001800 )
Phosphate-buffered Saline (PBS) (Corning, catalog number: 21-040-CV )
Methanol (Sigma-Aldrich, catalog number: 34860-1L-R )
Dichloromethane (Sigma-Aldrich, catalog number: 650463-1L )
HEPES buffer pH 7.4 (Sigma-Aldrich, catalog number: F7876 )
Negative control siRNA (Santa Cruz, catalog number: sc-37007 )
Positive CD98 siRNA (Santa Cruz, catalog number: sc-35033 )
Glucose (Sigma-Aldrich, catalog number: 49163 )
Turbofect^TM reagent (Thermo Fisher Scientific, catalog number: R0531 )
Potassium chloride (KCl, Millipore, catalog number: 7447-40-7 )
Quant-iT Ribogreen reagent (Invitrogen, catalog number: R11491 )
Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400-4 )
DiR dye (DiIC₁₈(7)) (Thermo Fisher Scientific, catalog number: D12731 )
Amicon^® ultra-15 centrifugal filter (MWCO 10,000) (Sigma-Aldrich, catalog number: UFC 9100 )
1% uranyl acetate solution (see Recipes)
5% glucose solution (see Recipes)

Equipment

Pipettes: 0.5-10 μl, 10-100 μl and 100-1,000 μl (Fisher Scientific, Eppendorf, catalog number: 13-684-251 )
Particle size analyzer (Brookhaven Instrument Corp., model: 90Plus )
Zetasizer (Malvern, model: Nano-ZS90 )
Atomic force microscopy instrument (Seiko Instrument Inc., model: SPA400 )
Transmission electron microscope (Carl Zeiss, model: LEO 906E )
Milli-Q advantage A10 water purification system (Millipore-sigma, catalog number: C10117 )
Centrifuge (Thermo Fisher Scientific, model: Sorvalis ST16R )
Ultracentrifuge (Beckman Coulter, model: Optima L-90K )
-80 °C freezer (So-Low, model: PV85-21 )
Sonicator (Branson, model: 3510 )
Vortexer (Scientific Industries, model: 200-SI0236 )
Rotary evaporator (Buchi, model: R-210 )
Vacuum pump (Buchi, model: V-700 )
Vacuum controller (Buchi, model: V-800 )
Heating bath (Buchi, model: B-491 )
Evaporating flask (Buchi, catalog number: Z402982 )
Liposome extruder (Avanti Polar Lipids, model: Mini extruder )
Microplate reader (BioTek, Model: Synergy2 )
IVIS in vivo imaging system (PerkinElmer, model: IVIS Spectrum Series )

Procedure

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Sung, J., Yang, C., Collins, J. F. and Merlin, D. (2020). Preparation and Characterization of Ginger Lipid-derived Nanoparticles for Colon-targeted siRNA Delivery. Bio-protocol 10(14): e3685. DOI: 10.21769/BioProtoc.3685.