Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Summary

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Abstract

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Introduction

Small interfering RNAs (siRNAs) are double-stranded RNA molecules that can suppress the expression of genes. In recent years, siRNAs have been developed as a new generation of biodrugs that show therapeutic potential for future use in clinical applications^1-5. However, the successful implementation of siRNA therapy remains a considerable challenge, due to degradation by nucleases, poor intracellular uptake, low transfection efficiency and inefficient release from the endosome/lysosome⁵. Many of these hurdles can be overcome by the development of delivery platforms, which can safely and efficiently deliver siRNA to diseased tissue. Compared to viral carriers, non-viral platforms provide several advantages, such as safety, low cost and ease of tailoring. In particular, cationic nanoparticles, such as polymers and lipids, have proved useful for siRNA delivery³.

Previously, we have developed a discoidal drug delivery system, termed the multistage vector (MSV). This platform is based on sequential stages, in which one vehicle is released from another. The first stage vehicle is a microparticle made from biodegradable porous silicon (pSi), while the second stage vehicles are nanoparticles loaded with drugs or contrast agents^6,7. The nanoparticles, which are embedded in the pSi material, are gradually released as the Si degrades⁸. A benefit of using Si particles is that the morphology and surface characteristics can easily be tailored to achieve optimal biodistribution and drug release. Recently, the successful use of the MSV platform for the delivery of siRNA liposomes to tumor tissue was shown in an ovarian and breast cancer mouse model^{9, 10}.

In this work, we have fabricated a universal delivery system for siRNA based on the principals of the MSV platform. The efficacy of this delivery system has previously been demonstrated using different siRNA molecules¹¹. The system is a polycation-functionalized porous silicon (PCPS) carrier, consisting of pSi grafted with polyethylenimine (PEI) and arginine (Arg). PEI can aid in forming electrostatic interactions with siRNA, while Arg and pSi can serve to reduce the toxicity of PEI, as previously demonstarted¹¹. In addition, the presence of PEI can assist in intracellular uptake and endosomal escape, while the pSi microparticles enable siRNA protection and sustained release. The pSi particles gradually degrade under physiological conditions, thereby resulting in the formation of Arg-PEI/siRNA nanoparticles (Figure 1), which have a distinct morphology and a narrow size distribution¹¹. For details regarding the stability of the PCPS/siRNA system, please refer to the study by Shen et al.¹¹. This PCPS platform differs from the conventional MSV, since the second stage nanoparticles are not initially present in the carrier, but are formed over time as the first-stage carrier degrades^{11, 12}. The siRNA loading efficiency, cytotoxicity and gene silencing efficiency of the PCPS system has been evaluated in vitro. Transfection efficiency was measured using siRNA against the ataxia telangiectasia mutated (ATM) oncogene, which is involved in DNA repair¹⁰. Previously, the suppression of ATM has been shown to decrease tumor growth in a breast cancer model¹⁰.

Protocol

1. PCPS Particle Preparation

1. Oxidize non-functionalized porous silicon particles in a 30% solution of hydrogen peroxide at 95 °C for 2 hr. Aminate the oxidized particles in 2% (3-​aminopropyl)​triethoxysilane solution in isopropyl alcohol for 2 days at 65 °C with gentle stirring.
2. Centrifuge the solution for 30 min at 18,800 x g and wash the particles twice in isopropyl alcohol and three times in ethanol, using brief sonication to suspend the pellet. Leave the particles in ethanol solution when performing step 1.3 and 1.4.
3. Add a known volume of particle suspension (e.g., 10 μl) to 10 ml isotone diluent and count the particles with a particle counting analyzer to determine the concentration of particles in the stock solution.
4. Activate the acid group of L-arginine (0.1 nmol) with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 0.1 nmol)/N-hydroxysuccinimide (NHS, 0.1 nmol) in 20 ml of ethanol for 2 hr at RT with gentle stirring.
5. Briefly sonicate the particle stock solution and add 1 billion particles to the L-arginine solution and leave the reaction for 18 hr at RT with gentle stirring.
6. Activate the first aspartic acid group of N-(tert-Butoxycarbonyl)-L-aspartic acid (Boc-Asp-OH, 1 nmol) with EDC (0.1 nmol)/NHS (0.1 nmol) in 20 ml of ethanol for 4 hr at 4 °C with gentle stirring.
7. Dissolve 50 mg polyethylenimine in 10 ml ethanol and add the solution to the Boc-Asp-OH mixture. Let the reaction proceed for 24 hr at RT with gentle stirring.
8. Activate the second aspartic acid group of the Boc-Asp-OH/PEI solution with EDC (0.1 nmol)/NHS (0.1 nmol) at 4 °C for 6 hr with gentle stirring.
9. To obtain PCPS particles, add the particle solution from step 1.5 into the Boc-Asp-OH/PEI solution from step 1.8. Allow the reaction to proceed for 18 hr at RT with gentle stirring.
10. Centrifuge the solution for 30 min at 18,800 x g and wash the particle solution three times with ethanol, using brief sonication to suspend the pellet.

2. PCPS Particle Characterization

1. Measure the size of the particles using a scanning electron microscope (SEM).
   1. Place a drop of particle suspension (10,000 particles/μl in ethanol) on a clean silica SEM sample stub and let dry at RT under vacuum.
   2. Measure SEM images at 8 kV with a 3–5 mm working distance using an in-lens detector.
2. Measure the zeta potential of the particles using a particle analyzer system.
   1. Mix 10 μl of particle suspension (10,000 particles/μl in ethanol) with 1 ml of 10 mM phosphate buffer (pH 7.4).
   2. Load the sample into folded capillary cells and measure the zeta potential according to the manufacturer’s instructions.

3. Loading of siRNA into PCPS Particles

1. Dry the PCPS particles (from PCPS particle preparation step 1.9) under vacuum O/N.
2. Add siRNA (4 μg) in nuclease-free water (20 μl) to the dried PCPS particles and sonicate briefly. Use the following particle to siRNA ratios: 2 × 10⁵ particles/0.2 μg siRNA, 4 × 10⁵ particles/0.2 μg siRNA, 6 × 10⁵ particles/0.2 μg siRNA, 8 × 10⁵ particles/0.2 μg siRNA, 10 × 10⁵ particles/0.2 μg siRNA and 12 × 10⁵ particles/0.2 μg siRNA.
3. Incubate for 3 hr at 4 °C on a shaker (1,000 rpm) to allow siRNA binding to the particles.

4. Optimization of siRNA/PCPS Particle Ratio

1. Add DNA loading dye to 20 μl of the PCPS/control siRNA particles with different particle to siRNA ratios (see loading of siRNA into PCPS particles).
2. Load the samples into a 2% agarose gel containing DNA gel stain.
3. Perform electrophoresis at a constant voltage of 120 V for 20 min in running buffer.
4. Analyze the gel with image acquisition and analysis software.

5. Release of siRNA from PCPS Particles

1. Mix 20 μl of the PCPS/control siRNA particles with different particle to siRNA ratios (see step 3) in sodium dodecyl sulfate (SDS, 2%) and let stand for 1 hr at RT.
2. Add DNA loading dye to the samples.
3. Load samples into a 2% agarose gel containing DNA gel stain.
4. Perform electrophoresis at a constant voltage of 120 V for 20 min in running buffer using DNA electrophoresis equipment and a power supply.
5. Analyze the gel with image acquisition and analysis software.

6. Confocal Microscopy of PCPS Particles

1. Add 5 μl of PCPS/fluorescent control siRNA particles (10 × 10⁵ particles/0.2 μg siRNA/20 μl) to a glass cover slip.
2. Visualize particle layers by confocal microscopy.

7. Characterization of Arg-PEI/control siRNA Nanoparticles

1. To degrade the silicon material and form Arg-PEI/control siRNA nanoparticles, add PCPS/siRNA particles (10 × 10⁶ PCPS particles/2 μg siRNA) to 100 μl of phosphate buffered saline and shake (1,000 rpm) at 37 °C for 2 days.
2. Centrifuge the sample for 30 min at 18,800 x g and collect the supernatant.
3. Measure the size of the formed Arg-PEI/siRNA nanoparticles with dynamic light scattering (DLS).
   1. Mix 10 μl of the supernatant with 1 ml 10 mM phosphate buffer (pH 7.4) and place into a plastic cuvette.
   2. Measure the size of the particles using a particle analyzer system according to the manufacturer’s instructions.
4. Determine the size and morphology of the formed Arg/PEI-siRNA nanoparticles with atomic force microscopy.
   1. Take 10 µl of the supernatant and place on a silicon wafer.
   2. Visualize the particles with atomic force microscopy (AFM).

8. Cell Culture

1. Culture MDA-MB-231 human breast cancer cells in cell culture media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 5% CO₂, 95% humidity and 37 °C.

9. Confocal Microscopy of Live Cells with PCPS Particles

1. Plate cells in 2-well culture slides at a seeding density of 3 x 10⁵ cells/well for 24 hr.
2. Add PCPS particles loaded with fluorescent control siRNA (10 × 10⁵ particles/0.2 μg particle to siRNA ratio, 50 nM siRNA) to cells.
3. Record a movie of the cells with a confocal microscope (supplied with a chamber, 5% CO₂, 95% humidity and 37 °C) for 12 hr following particle exposure.

10. Confocal Microscopy of Fixed Cells with PCPS Particles

1. Plate cells in 2-well culture slides at a seeding density of 3 x 10⁵ cells/well for 24 hr.
2. Add PCPS particles loaded with fluorescent control siRNA (10 × 10⁵ particles/0.2 μg particle to siRNA ratio, 50 nM siRNA) to cells and incubate for 1 day, 7 days and 10 days.
3. Wash the cells twice with phosphate buffered saline and then fix them with 4% paraformaldehyde solution for 10 min.
4. Wash the cells with phosphate buffered saline.
5. Permeabilize the cells with 0.1% octyl phenol ethoxylate for 10 min and then wash them three times with phosphate buffered saline.
6. Block the cells with albumin from bovine serum (10 mg/ml) in phosphate buffered saline for 10 min at RT with gentle stirring.
7. To visualize filamentous actin, incubate the cells with fluorescently labeled phalloidin (1 μl/40 μl blocking solution) for 20 min at RT with gentle stirring and then wash in phosphate buffered saline.
8. Remove the slides from the frame and add antifade reagent with 4',6-diamidino-2-phenylindole (DAPI) to visualize the nucleus.
9. Add a cover glass on top and take images of the cells with confocal microscopy.

11. Flow Cytometry of Cells with PCPS/fluorescent Control siRNA Particles

1. Plate cells in a 6-well plate at a seeding density of 3 x 10⁵ cells/well for 24 hr.
2. Add PCPS particles loaded with fluorescent control siRNA (10 × 10⁵ particles/0.2 μg particle to siRNA ratio, 50 nM siRNA) to the cells and incubate for 24 hr.
3. Wash the cells with phosphate buffered saline and scrape them with a cell scraper.
4. Store the cells in phosphate buffered saline with 2% fetal bovine serum prior to analysis. Use untreated cells as a negative control.
5. Perform flow cytometry.

12. Cell Viability of Cells with PCPS Particles and PCPS/control siRNA Particles

1. Plate cells in a 96-well plate at a cell density of 3 × 10³ cells/well for 24 hr.
2. Treat the cells with PCPS particles (1.5 × 10⁵/well and 6 × 10⁵/well) or PCPS/control siRNA particles (10 × 10⁵ particles/0.2 μg particle to siRNA ratio, 10 nM and 100 nM siRNA) for 48 hr and 72 hr. Use untreated cells and cells treated with phosphate buffered saline (same volume as the added particles) as controls. Assay each sample in triplicate.
3. Perform a cell proliferation assay according to the manufacturer’s instructions.
4. Represent data as the mean ± standard deviation.

13. Western Blot of Cells with PCPS/ATM Mutated siRNA Particles

1. Plate cells in a 6-well plate at a cell density 2 × 10⁵ cells/well for 24 hr.
2. Incubate the cells with PCPS/control siRNA (50 nM) particles or PCPS/ATM siRNA (50 nM) particles for 72 hr. Use untreated cells as a control.
3. Lyse the cells using a protein extraction reagent supplemented with a protease inhibitor cocktail.
4. Centrifuge the cell lysates for 10 min at 14,000 x g and recover the supernatant.
5. Determine the protein concentration with a protein quantification assay according to the manufacturer’s instructions.
6. Add sample loading buffer (with 5 μl 2-mercaptoethanol/ml buffer) to the samples and heat them for 6 min at 99 °C.
7. Load the protein samples (20 μg/μl) in a 12% SDS-polyacrylamide gel in running buffer and perform polyacrylamide gel electrophoresis (1 hr, 120 V) using electrophoresis equipment and a power supply.
8. Transfer the gel in transfer buffer (with 20% methanol) to a nitrocellulose membrane (1 hr, 100 V) using electrophoresis equipment and a power supply.
9. Block the membrane with 5% dry milk for 1 hr.
10. Incubate the membrane with the ATM primary antibody (from rabbit) in blocking solution (from step 13.9) at a 1:1,000 dilution O/N.
11. Wash the membrane with phosphate buffered saline containing 0.1% polyethylene glycol sorbitan monolaurate and then incubate it with the secondary antibody (anti-rabbit) in blocking solution (from step 13.9) at a 1:2,500 dilution for 1 hr.
12. Wash the membrane with phosphate buffered saline containing 0.1% polyethylene glycol sorbitan monolaurate and detect the protein bands with Western blot detection reagent using image acquisition and analysis software.
13. For the loading control, wash the membrane and repeat steps 13.9-13.12 using a β-actin primary antibody (from mouse, 1:10,000 dilution) and a secondary antibody (anti-mouse 1:4,000 dilution).

Results

This protocol describes the use of a non-viral delivery system for safe and efficient siRNA transfection. The SEM results reveal that the PCPS particles are cylindrical in shape and have a diameter of 2.6 μm (Figure 2A). The particles are positively charged with a zeta potential of approximately +8.21 (Figure 2B), thereby enabling electrostatic binding with negatively charged nucleotides. Confocal images of different layers of the PCPS particles demonstrate that fluorescent control siRNA...

Discussion

This protocol describes a method for the successful delivery and transfection of siRNA into cells. In particular, the delivery of siRNA is achieved by using a multifunctional platform consisting of polycation-functionalized pSi particles. The use of siRNA therapy has great potential, e.g., cancer treatment, as various oncogenes can be targeted with high specificity. Therefore, there exists a demand to develop siRNA delivery vehicles, which can mitigate the challenges of siRNA therapy. In conclusion, we have outl...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Polyethylenimine (PEI), branched	Sigma-Aldrich	408727	Average molecular weight ~25,000 Da
L-Arginine	Sigma-Aldrich	A5006	Reagent grade, ≥98%
Boc-Asp-OH	Sigma-Aldrich	408-468	99%
Hydrogen peroxide solution	Sigma-Aldrich	216763	30 wt. % in H2O
Sulfuric acid	Sigma-Aldrich	339741	100.00%
Isopropyl alcohol	Sigma-Aldrich	W292907	≥99.7%
Ethanol	Sigma-Aldrich	459844	≥99.5%
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)	Sigma-Aldrich	03449	≥99%
Albumin from bovine serum	Sigma-Aldrich	A7030-10G	Blocking agent
Ataxia-telangiectasia mutated siRNA	Sigma-Aldrich		Designed in-house
Tris Acetate-EDTA buffer	Sigma-Aldrich	T9650	For DNA agarose gel electrophoresis
Penicillin-Streptomycin	Sigma-Aldrich	P4333
Fetal Bovine Serum	Sigma-Aldrich	F2442
TWEEN 20	Sigma-Aldrich	P1379	Polyethylene glycol sorbitan monolaurate
2-Mercaptoethanol	Sigma Aldrich	M6250	For Western blot
Sodium dodecyl sulfate	Sigma-Aldrich	L3771
Sodium phosphate monobasic	Sigma-Aldrich	71496	For making phosphate buffer
Sodium phosphate dibasic	Sigma-Aldrich	71640	For making phosphate buffer
Anti-Mouse IgG	Sigma-Aldrich	A4416	Secondary antibody (anti-mouse) for Western blot
N-Hydroxysuccinimide (NHS)	Sigma-Aldrich	130672	98%
CELLSTAR 96W Microplate Tissue Culture Treated Clear w/ Lid	Greiner Bio-One	655182	96-well plate
10x Tris-Glycine Liquid	Li-Cor	928-40010	Transfer buffer for Western blot
Paraformaldehyde solution 4% in PBS	Santa Cruz	Sc-281692	Fixation of cells
CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS)	Promega	G5421	Proliferation assay
Phosphate buffered saline	Fisher Scientific	BP399-500	10x Solution
Corning cellgro Dulbecco's Modification of Eagle's (Mod.)	Fisher Scientific	MT-15-017-CM	Cell culture media, 1x solution
Triton X-100	Fisher Scientific	AC21568-2500	Octyl phenol ethoxylate, permeabilization agent
Cover glass	Fisher Scientific	12-530C
Methanol	Fisher Scientific	A412-1	For Western blot transfer buffer
Plastic Cuvettes	Fisher Scientific	14-377-010	For size measurements using Zetasizer Nano ZS
Molecular BioProducts RNase AWAY Surface Decontaminant	Fisher Scientific	14-754-34	Spray for removing RNAse contamination
Agarose	Fisher Scientific	BP165-25	Low melting point, for running RNA samples
ProLong Gold Antifade Reagent with DAPI	Invitrogen	P36935	Antifade reagent with DAPI, nucelus detection
Alexa Fluor 488 Phalloidin	Invitrogen	A12379	Dissolve 300 units in 1.5 ml methanol, detection of filamentous actin
SYBR Safe DNA Gel Stain	Invitrogen	S33102	Visualization of RNA
Negative Control siRNA	Qiagen	1022076	Control siRNA
AllStars Neg. siRNA AF 555	Qiagen	1027294	Fluorescent control siRNA
Cell scraper	Celltreat	229310
BioLite Multidishes and Microwell Plates	Thermo Scientific	130184	6-well plate
Pierce LDS Sample Loading Buffer (4x)	Thermo Scientific	84788	Sample loading buffer for Western blot
Pierce BCS Protein Assay Kit	Thermo Scientific	23227	Protein quantification assay
Halt Protease Inhibitor Single-Use Cocktails (100x)	Thermo Scientific	78430	Protease inhibitor cocktail, use at 1x
M-PER Mammalian Protein Extraction Reagent	Thermo Scientific	78501	Protein extraction reagent
Sorvall Legend Micro 21R	Thermo Scientific	75002440	Centrifuge
Beta Actin Antibody	Thermo Scientific	MA1-91399	β-actin primary antibody (from mouse) for western blot
6x TriTrack DNA Loading Dye	Thermo Scientific	R1161	DNA loading dye
Nuclease-Free Water	Life Technologies	AM9938
Non-Fat Dry Milk	Lab Scientific	M0841	For Western blot
2-well BD Falcon culture slides	BD Biosciences	354102	2-well culture slides
Amersham ECL Western blot detection reagent	GE Healthcare Life Sciences	RPN2106	Western blot detection reagent
BA Membranes	GE Healthcare Life Sciences	10402096	Nitrocellulose membrane for Wester blot
ATM (D2E2) Rabbit mAb	Cell Signaling	2873S	ATM primary antibody (from rabbit) for Western blot
Anti-rabbit IgG, HRP-linked Antibody	Cell Signaling	7074	Secondary antibody (anti-rabbit) for Western blot
Folded capillary cells	Malvern	DTS 1061	For zeta potentail measurements using Zetasizer Nano ZS
MDA-MB-231 cell line	ATCC	HTB-26	Mammary Gland/Breast
12% Mini-PROTEAN TGX Gel	Bio-rad	456-1043	For Western blot
Biorad PowerPac HC	Bio-rad	164-5052	Power supply for electrophoresis
10x Tris/Glycine/SDS Buffer	Bio-rad	161-0732	Running buffer for Western blot
Wide Mini-Sub Cell GT Cell	Bio-rad	170-4405	Electrophoresis equipment for DNA agarose gel
Mini-PROTEAN Tetra cell	Bio-rad	165-8000	Electrophoresis equipment for Western blot
ChemiDoc XRS+ System with Image Lab Software	Bio-rad	170-8265	Image acquisition and analysis software for gels and blots
4" (10 cm) dia., 5 x 7 mm diced Silicon Wafer	Ted Pella	16007	Silicon waferfor scanning electron microscopy and atomic force microscopy
Thermomixer R	Eppendorf	22670107	Shaker
Isoton II diluent	Beckman Coulter	8546719	Isoton diluent
Multisizer 4 Coulter Counter	Beckman Coulter	A63076	Particle counting analyzer
Non-functionalized porous silicon particles	Microelectronics Research Center, University of Texas at Austin		Dicoidal shape. 2.6 μm (diameter) x 0.7 μm (height), provided in isopropyl alcohol
Zetasizer Nano ZS	Malvern		Particle analyzer system for size and zeta potential
Scanning Electron Microscope	FEI		Particle size and shape
Atomic Force Microscope	Bruker		Particle size and shape
Fluo ViewTM 1000 Confocal Microscope	Olympus		Visualization of fixed and live cells