Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery

Summary

We demonstrate the synthesis of fusogenic porous silicon nanoparticles for effective in vitro and in vivo oligonucleotide delivery. Porous silicon nanoparticles are loaded with siRNA to form the core, which is coated by fusogenic lipids through extrusion to form the shell. Targeting moiety functionalization and particle characterization are included.

Abstract

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic porous silicon nanoparticles (F-pSiNPs) have recently demonstrated high in vivo gene silencing efficacy due to its high oligonucleotide loading capacity and unique cellular uptake pathway that avoids endocytosis. The synthesis of F-pSiNPs is a multi-step process that includes: (1) loading and sealing of oligonucleotide payloads in the silicon pores; (2) simultaneous coating and sizing of fusogenic lipids around the porous silicon cores; and (3) conjugation of targeting peptides and washing to remove excess oligonucleotide, silicon debris, and peptide. The particle’s size uniformity is characterized by dynamic light scattering, and its core-shell structure may be verified by transmission electron microscopy. The fusogenic uptake is validated by loading a lipophilic dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), into the fusogenic lipid bilayer and treating it to cells in vitro to observe for plasma membrane staining versus endocytic localizations. The targeting and in vivo gene silencing efficacies were previously quantified in a mouse model of Staphylococcus aureus pneumonia, in which the targeting peptide is expected to help the F-pSiNPs to home to the site of infection. Beyond its application in S. aureus infection, the F-pSiNP system may be used to deliver any oligonucleotide for gene therapy of a wide range of diseases, including viral infections, cancer, and autoimmune diseases.

Introduction

Gene therapy modulates specific gene expression to obtain a therapeutic outcome. Numerous tools for gene modulation have been discovered and studied, including ribonucleic acid interference (RNAi) using oligonucleotides (e.g., short interfering RNA (siRNA)^1,2, microRNA (miRNA)^3,4), DNA plasmids^5,6, nucleases (e.g., zinc finger, TALENS)^7,8, and CRISPR/Cas9 systems^9,10. While each....

Protocol

1. Synthesis of porous silicon nanoparticles (pSINPs)

CAUTION: Always use caution when working with hydrofluoric acid (HF). Follow all safety guides according to its safety data sheet (SDS), handle any HF-containing chemicals in a fume hood, and wear appropriate personal protective equipment (PPE; double gloves with butyl gloves on the outside, butyl apron with lab coat underneath, face shield with safety goggles underneath). All universities and R&D labs require specific training on HF safety prior to usage. Do not attempt to work with HF without pre-approval of your local lab safety coordinator, as additional safety measures not describ....

Results

A successful synthesis of fusogenic pSiNPs should produce a homogenous, slightly opaque solution (Figure 3a). Failure to optimize the ratio and concentration of pSiNPs : siRNA : CaCl₂ may lead to aggregation upon loading (Figure 3b). As the particles are extruded through 200 nm membranes, the average hydrodynamic diameter of the fusogenic pSiNPs measured by DLS should be approximately 200 nm, and the average zeta-poten.......

Discussion

Synthesis of porous silicon nanoparticles is shown in Figure 5. The critical step in the synthesis of fusogenic pSiNPs is in the loading step (step 3). If the fusogenic nanoparticles are aggregating post-synthesis (Figure 3), the reason may be due to the following: (1) calcium chloride stock was not homogenously prepared, thus step 3.1.2 must be carefully followed or refined; or (2) the ratio of pSiNP : siRNA : CaCl₂ or the concentration of one or mor.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)	Avanti Polar Lipids	850345P	Powder
1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP)	Avanti Polar Lipids	890890P	Powder
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG(2000) Maleimide)	Avanti Polar Lipids	880126P	Powder
Aluminum foil	VWR International, LLC	12175-001
Calcium chloride (CaCl2)	Spectrum	C1977	Anhydrous, Pellets
Chloroform	Fisher Scientific	C6061
Computer	Dell	Dimension 9200	Any computer with PCI card slot is acceptable
Dil Stain (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate ('DiI'; DiIC18(3)))	Life Technologies	D3911
Ethanol (EtOH)	UCSD Store	111	200 Proof
Hydrofluric acid (HF)	VWR International, LLC	MK264008	Purity: 48%
Keithley 2651a Sourcemeter	Keithley	2651A
LabVIEW	National Instruments		Sample program available at: http://sailorgroup.ucsd.edu/sofware/
LysoTracker Green DND-26	Thermo Fisher Scientific	L7526
Liposome extrusion set with heating block	Avanti Polar Lipids	610000
Microcon-30kDa Centrifugal Filter Unit	EMD Millipore	MRCF0R030
O-ring	ChemGlass	CG-305-220
Phosphate-buffered saline (PBS)	Thermo Fisher Scientific	10010-049
Platinum coil	VWR International, LLC	AA10285-BU
Potassium hydroxide (KOH)	Fisher Scientific	P250-3
Silicon wafer	Siltronix	Custom order
siRNA	Dharmacon	Custom order	IRF5, sense 5’-dTdT-CUG CAG AGA AUA ACC CUG A-dTdT-3’ and antisense 5’-dTdT UCA GGG UUA UUC UCU GCA G dTdT-3’
Sonicator	VWR International, LLC	97043-960
Targeting peptide (CRV)	CPC Scientific	Custom order	sequence CRVLRSGSC; made cyclic by a disulfide bond between the side chains of the two cysteine residues
Teflon etch cell	Interface Performance Materials, Inc.	Custom order
UltraPure DNase/RNase-Free Distilled Water	Thermo Fisher Scientific	10977015