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 ...

This protocol describes the synthesis of a nanoparticle system that is able to silence genes in vivo by delivering siRNAs with high efficiency. By simply exchanging the targeting peptide and the siRNA payload, the fusogenic nanoparticles may be used as a potential therapeutic formulation for diseases that require in vivo gene modulation. We have previously published results using the fusogenic nanoplatform against gram-positive bacterial infections by silencing a gene that encodes for inflammation in macrophages to terminate chronic inflammation.Prior to performing this procedure, assemble a Teflon etch cell containing a silicon wafer in a three-to-one hydrofluoric acid solution. Run an alternating current of a square waveform with a low current density of 50 milliamperes per centimeter squared for 0.6 seconds and a high current density of 400 milliamperes per centimeter squared for 0.36 seconds repeated for 500 cycles. Once the etching is finished, remove the cell from the circuit, and carefully rinse out the hydrofluoric acid solution using a syringe.Then, rinse three times with ethanol. To lift off the porous layer from the silicon wafer, first fill the well of the etch cell with 10 milliliters of a one-to-29 hydrofluoric acid solution. Then, run a constant of 3.7 milliamperes per centimeter squared for 250 seconds.Once the etching is finished, remove the cell from the circuit. The porous silicon layer may have visible ripples, indicating detachment from the crystalline silicon wafer. Gently wash out the hydrofluoric acid solution, and rinse three times each with ethanol and water.Using a pipette tip, firmly crack the circumference of the porous silicon layer for a complete detachment. Using ethanol, collect the porous silicon fragments, or chips, from the Teflon etch cell into a weighing boat. Then, transfer the chips to a glass vial for storage.Next, replace the ethanol in the glass vial containing the porous silicon chips with two milliliters of RNase-free water. Firmly cap and seal the vial using Parafilm. Place the glass vial in a sonicator bath and suspended such that the volume of porous silicon chips are completely submerged below the surface of the water bath.To prevent significant water loss during sonication, place a volumetric flask filled with water, inverted so that the opening of the flask touches the surface of the water bath. Then, sonicate the porous silicon chips for 12 hours at 35 kilohertz with an RF power of 48 watts. After sonication, place the glass vial on a flat surface for one hour to allow the larger particles to settle at the bottom.Then, collect the supernatant using a pipette. In a glass vial, add 72.55 microliters of DMPC solution, 15.16 microliters of DSPE-PEG solution, and 19.63 microliters of DOTAP solution, and mix by pipetting. Place the vial in a fume hood with a loose cap to allow the solvent to evaporate overnight.The dried film will be a cloudy, hard, gel-like substance at the bottom of the vial. Next, place a microcentrifuge tube containing 150 microliters of porous silicon nanoparticles in a previously prepared iced sonication bath. Under a 15-minute ultrasonication, gently pipette 150 microliters of siRNA and 700 microliters of two-molar calcium chloride into the porous silicon nanoparticles.Remove the tube containing one milliliter of siRNA-loaded calcium-coated porous silicon nanoparticles from the sonicator. Fill a wide beaker with deionized water. Soak a polycarbonate membrane and four filter supports by floating them on the water surface.Then, assemble a liposome extrusion kit by following the manufacturer's instructions. Next, hydrate the dried lipid film in the glass vial with one milliliter of the siRNA-loaded calcium-coated porous silicon nanoparticles. Pipette until all lipids have lifted from the bottom of the vial and a cloudy, homogenous solution is observed.Add a magnetic stirring bar to the glass vial, and place the vial on a hot plate to heat the particles to 40 degrees Celsius while magnetically stirring the solution for 20 minutes. Following this, attach an empty syringe to one side of the extruder. Then, fill another syringe with one milliliter of the lipid-coated porous silicon nanoparticle solution, and attach the syringe to the other side of the extruder.Begin the extrusion by pushing the piston in slowly to push the particles from one syringe, through the polycarbonate membrane, and into the other syringe. Repeat 20 times. Then, collect the extruded particles into a microcentrifuge tube.Prepare a peptide stock with a one-milligram-per-milliliter peptide concentration in RNase-free water. In the microcentrifuge tube containing the fusogenic lipid-coated porous silicon nanoparticles, add 100 microliters of the peptide stock, and pipette gently. Keep the tube static at room temperature for 20 minutes.To remove excess peptide or siRNA and other excipients, wash in a 30-kilodalton centrifugal filter by spinning at 5, 000 times g at room temperature for one hour. Centrifuge two more times with one milliliter of PBS under the same settings. Following centrifugation, resuspend the final peptide-conjugated fusogenic lipid-coated porous silicon nanoparticles in PBS at the desired concentration.The particles can be aliquoted and stored at minus 80 degrees Celsius for at least 30 days. A successful synthesis of fusogenic porous silicon nanoparticles should produce a homogenous, slightly opaque solution that may be stored for 30 days and thawed without causing structural changes. Failure to optimize the ratio and concentration, as well as repeated freeze-thaw cycles may lead to aggregation upon loading.As the particles are extruded, the average hydrodynamic diameter of the fusogenic porous silicon nanoparticles measured by dynamic light scattering should be approximately 200 nanometers and the average zeta-potential approximately positive seven millivolts. After surface modification with targeting peptides, the overall diameter should be less than 230 nanometers, and the average zeta-potential decreases to minus 3.4 millivolts. Fusion may be confirmed by labeling the fusogenic lipids with the lipophilic fluorophore DiI and observing the in vitro localization using confocal microscopy.Successful fusion is observed when the fusogenic porous silicon nanoparticle's lipids transfer the DiI to the plasma membrane and are localized independent of lysosomes. Unsuccessful fusion will show the DiI localization within the cell's cytoplasm and co-localization with lysosomes. This fusogenic nanoparticle system is now being used for therapeutic applications in not only bacterial infections but also in cancer adjuvant therapy and cancer immunotherapy.This protocol may be further optimized to load more than siRNAs. The fusogenic nanoparticle system has shown potential in loading and delivering larger anionic payloads, such as mRNAs and CRISPR/Cas9 complexes.

synthesis-functionalization-characterization-fusogenic-porous-silicon

Research

JoVE Journal

Engineering

7.4K visualizações.  University of California, San Diego. Este protocolo descreve a síntese de um sistema de nanopartículas que é capaz de silenciar genes in vivo, fornecendo siRNAs com alta eficiência. Ao simplesmente trocar o peptídeo direcionado e a carga de siRNA, as nanopartículas fusogênicas podem ser usadas como uma formulação terapêutica potencial para doenças que requerem modulação genética in vivo. Publicamos resultados anteriormente usando a nanoplataforma fusogênica contra infecções bacterianas gram-positivas silenciando...