Name: synthesis, functionalization, and characterization of fusogenic porous silicon nanoparticles for oligonucleotide delivery
Uploaded: 2019-04-16T14:45:00
Description: Watch this Scientific Journal Video about Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery at JoVE.com

This work is supported by National Institutes of Health through contract # R01 AI132413-01.

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&#38;D labs require specific training on HF safe...

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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...

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 plasmids5,6, nucleases (e.g., zinc finger, TALENS)7,8, and CRISPR/Cas9 systems9,10. While each tool&#8217;s mechanism of action differs, all of the tools must reach the cell&#8217;s cytoplasm or the nucleus to be active. As such, while these tools have proven to induce significant effect in modulating gene expression in vitro, the in vivo efficacy suffers from extracellular and intracellular obstacles. Due to the fact that the tools are of biological origin, many enzymes and clearance systems exist in our body that have the ability to degrade or remove the foreign molecules11. Even in the case that the tools reach the target cell, they suffer from endocytosis; a mode of cellular uptake that encapsulates and traps the tools in acidic stomach-like vesicles that degrade or expel the tools out of the cell. In fact, studies have shown that lipid nanoparticles are endocytosed via macropinocytosis, from which approximately 70% of the siRNA are exocytosed from the cells within 24h of uptake12,13. The majority of the remaining siRNA are degraded through the lysosomal pathway, and ultimately only 1-2% of the siRNA that initially enters the cell with the nanoparticles achieve endosomal escape to potentially undergo RNAi13,14.
We have recently developed fusogenic porous silicon nanoparticles (F-pSiNPs) that have an siRNA-loaded core composed of porous silicon nanoparticles, and a fusogenic lipid shell15. The F-pSiNPs present three major advantages over other conventional oligonucleotide delivery systems: (1) a fusogenic lipid coating which enables the particles to bypass endocytosis and deliver the entire payload directly in the cell cytoplasm (versus the 1-2% achieved by endocytosed particles13,14) (Figure 1); (2) high mass loading of siRNA in the pSiNPs (&#62;20 wt% compared to 1-15 wt% by conventional systems)15, which rapidly degrade in the cytoplasm (once the core particles shed the lipid coating via fusogenic uptake) to release the siRNA; and (3) targeting peptide conjugation for selective homing to desired cell types in vivo. &#160;
The F-pSiNP system has demonstrated significant gene silencing efficacy (&#62;95% in vitro; &#62;80% in vivo) and subsequent therapeutic effect in a fatal mouse model of S. aureus pneumonia; the results of which were published previously15. However, the complex structure of the F-pSiNP system requires delicate handling and fine-tuned optimization to generate uniform and stable nanoparticles. Thus, the purpose of this work is to present a thorough protocol, as well as optimization strategies for the synthesis, functionalization, and characterization of F-pSiNPs to be used in targeted delivery of siRNAs for potent gene silencing effect.

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synthesis, functionalization, and characterization of fusogenic porous silicon nanoparticles for oligonucleotide delivery

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&#8217;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&#39;-dioctadecyl-3,3,3&#39;,3&#39;-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.

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...

Watch this Scientific Journal Video about Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery at JoVE.com

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

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.

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic ...

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