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 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 described here are required.
<ol>
	<li>Preparation of the etching solutions
	<ol>
		<li>To make the 3:1 HF solution for etching (used in Steps 1.3 and 1.4), fill a plastic graduated cylinder with 30 mL of aqueous 48% HF and 10 mL of absolute ethanol (EtOH). The solution must be contained in high density plastics (e.g., HDPE), as the HF dissolves glass.</li>
		<li>To make a 1:29 HF solution for lift-off (used in Step 1.5), fill a plastic graduated cylinder with 1 mL of aqueous 48% HF and 29 mL of absolute ethanol. The solution must be contained in high density plastics (e.g., HDPE), as the HF dissolves glass.</li>
		<li>Make the 1 M KOH solution in 10% EtOH for excess pSi dissolution (used in Steps 1.3 and 1.5).</li>
	</ol>
	</li>
	<li>Setting up the etch cell 
	<ol>
		<li>In a Teflon etch cell with a 8.6 cm2 etch well (area is calculated by measuring the diameter of the silicon surface available for etching within the O-ring, and calculating the area by A = &#960;r2), place in descending order (Figure 2a): (1) the Teflon cell top; (2) an O-ring; (3) a quarter piece of the single crystal (1 0 0)-oriented p++-type silicon wafer cut into quarters (using a diamond cutter); (4) aluminum foil; and (5) the Teflon cell base with screws gently tightened to prevent leakage.</li>
		<li>Fill the well with EtOH. Take a wipe and insert into the crevice between the Teflon cell top and base. If the wipe is dry upon removal, the etch cell is sealed. If wet, the cell is leaking, and tighten the screws further until sealed.</li>
	</ol>
	</li>
	<li>Electropolishing the silicon wafer
	<ol>
		<li>Bring the assembled etch cell into a fume hood.</li>
		<li>Fill the well with 10 mL of 3:1 HF solution.</li>
		<li>Connect the positive lead to the aluminum foil (electrode) from the etch cell and connect the negative lead to the platinum coil (counter-electrode) immersed in the 3:1 HF solution of the etch cell to complete the circuit (Figure 2b).</li>
		<li>Run a constant current at 50 mA cm-2 for 60 s. Once etch is finished, remove the cell from the circuit, and rinse out the 3:1 HF carefully using a syringe. Rinse with EtOH three times.</li>
		<li>Dissolve away the etched layer by filling the well slowly with 10 mL of 1 M KOH. Wait until the bubbling subsides.</li>
		<li>Rinse out the KOH with water three times, and then with EtOH three times.</li>
	</ol>
	</li>
	<li>Electrochemically etching porous layers into silicon wafer
	<ol>
		<li>Run an alternating current of a square waveform, with lower current density of 50 mA/cm2 for 0.6 s and high current density of 400 mA/cm2 for 0.36 s repeated for 500 cycles.</li>
		<li>Once etch is finished, remove the cell from the circuit, and rinse out the 3:1 HF carefully using a syringe. Rinse with EtOH three times.</li>
	</ol>
	</li>
	<li>Lifting-off the porous layer from the silicon wafer
	<ol>
		<li>Fill the well with 10 mL of 1:29 HF solution.</li>
		<li>Run a constant of 3.7 mA/cm2 for 250 s. Once etch is finished, remove the cell from the circuit. The pSi layer may have visible ripples indicating detachment from the crystalline silicon wafer. Gently wash out the 1:29 HF solution and rinse with EtOH three times, then with water three times.</li>
		<li>Using a pipette tip, firmly crack the circumference of the pSi layer for complete detachment. Using EtOH, collect the pSi fragments (chips) from the Teflon etch cell into a weighing boat. Transfer the chips to a glass vial. The pSi chips may be kept at room temperature in EtOH for over 6 months for storage.</li>
		<li>Dissolve away any remaining porous silicon on the wafer by filling the well slowly with 10 mL of 1 M KOH. Wait until the bubbling subsides.</li>
		<li>Rinse out the KOH with water three times, and then with EtOH three times.</li>
		<li>Repeat Steps 1.3-1.5 until the entire wafer thickness has been etched.</li>
	</ol>
	</li>
	<li>Sonicating porous layers for nanoparticle formation 
	<ol>
		<li>Replace the solvent of the glass vial containing pSi chips from EtOH to 2 mL of DI water.</li>
		<li>Firmly close the cap, and seal using parafilm.</li>
		<li>Place the glass vial in a sonicator bath and suspended such that the volume of pSi chips is completely submerged below the surface.</li>
		<li>Sonicate for 12 h at 35 kHz and RF power of 48W. 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.</li>
		<li>Place the glass vial on a flat surface for 1 h to allow larger particles to settle at the bottom. Collect the supernatant using a pipette; the suspension contains sub-100 nm pSiNPs.</li>
	</ol>
	</li>
</ol>
2. Preparation of fusogenic lipid film
<ol>
	<li>Preparation of the lipid stock solutions
	<ol>
		<li>In a fume hood, make 10 mg/mL stocks of lipids by hydrating DMPC, DSPE-PEG-MAL and DOTAP lipids in chloroform. These stock solutions may be kept at -20 &#176;C for up to 6 months under tight parafilm seal.</li>
	</ol>
	</li>
	<li>Making the fusogenic lipid film 
	NOTE: The fusogenic composition is made of the lipids, DMPC, DSPE-PEG, and DOTAP, at the molar ratio of 76.2:3.8:20 and 96.2:3.8:0, respectively.
	<ol>
		<li>In a glass vial, mix 72.55 &#956;L of DMPC, 15.16 &#956;L of DSPE-PEG, and 19.63 &#956;L of DOTAP by pipetting.</li>
		<li>Optional: For fluorescent labelling of the lipid coating, additionally add 20 &#181;L of DiI dissolved in EtOH at a concentration of 1 mg/mL.</li>
		<li>Place the vial in a fume hood with a loose cap to allow the chloroform to evaporate overnight. The dried film will be a cloudy hard gel-like substance at the bottom of the vial.</li>
	</ol>
	</li>
</ol>
3. Loading and sealing of siRNA in pSiNPs
<ol>
	<li>Preparation of the calcium chloride loading stock
	<ol>
		<li>Dissolve 1.11 g of CaCl&#173;2 in 10 mL of RNAse-free water to make a 2 M CaCl2 solution.</li>
		<li>Centrifuge the solution at &#62; 10,000 x g for 1 min to settle the aggregates and collect the supernatant using a pipette. Alternatively, filter the solution through a 0.22 &#181;m filter.</li>
	</ol>
	</li>
	<li>Preparation of the siRNA loading stock
	<ol>
		<li>Hydrate or dilute the siRNA to 150 &#181;M in RNAse-free water. This stock solution may be aliquoted and kept frozen at -20 &#176;C for at least 30 days given that it does not undergo frequent freeze-thaw cycles.</li>
	</ol>
	</li>
	<li>Loading the siRNA in pSiNPs with calcium chloride
	<ol>
		<li>Prepare an iced sonication bath.</li>
		<li>Under 15 min ultrasonication, gently pipette 150 &#181;L of siRNA and 700 &#181;L of 2 M CaCl2 into 150 &#181;L of pSiNP. Make sure to leave the lid of the microcentrifuge tube open for gas generation.</li>
		<li>Remove the tube containing 1 mL of siRNA-loaded calcium coated pSiNPs from the sonicator.</li>
	</ol>
	</li>
</ol>
4. Coating siRNA-loaded pSiNPs with fusogenic lipids
<ol>
	<li>Preparation of the liposome extrusion kit
	<ol>
		<li>Fill a wide beaker with DI water. Soak 1 polycarbonate membrane (200 nm pores), and 4 filter supports by floating them on water surface. Assemble the liposome extrusion kit by following manufacturer&#8217;s instructions</li>
	</ol>
	</li>
	<li>Hydrating the lipid film with siRNA-loaded pSiNps
	<ol>
		<li>Hydrate the dried lipid film in the glass vial with 1 mL of of siRNA-loaded calcium coated pSiNPs obtained from step 3.3.3. Pipette until all lipids film have lifted from the bottom of the vial and have mixed into a cloudy homogenous solution.</li>
		<li>Place a magnetic stirring bar in the glass vial, and place the vial onto a hot plate to heat the particles to 40 &#176;C (or high enough above the lipids&#8217; phase transformation temperature to maintain the lipids in the more fluidic liquid crystal phase) while magnetically stirring the solution for 20 min.</li>
	</ol>
	</li>
	<li>Extruding the lipid-coated pSiNPs for size control
	<ol>
		<li>Aspirate the 1 mL of lipid-coated pSiNP-siRNA solution in the extrusion syringe. Insert an empty syringe on one side of the extruder, and the filled syringe on the other end.</li>
		<li>Begin extrusion by pushing the piston in slowly to push the particles from one syringe, through the polycarbonate membrane, and into the other. Repeat 20 times. If the piston is stuck, the filter and membrane may be clogged. Disassemble the extruder and replace the membrane and filter supports. If the problem persists, dilute the particles until clogging is manageable.</li>
		<li>Collect the extruded particles into a microcentrifuge tube.</li>
	</ol>
	</li>
</ol>
5. Conjugation of targeting peptides
<ol>
	<li>Conjugating targeting peptide to lipid coating
	<ol>
		<li>Prepare the peptide stock with a 1 mg/mL peptide concentration in RNAse-free water.</li>
		<li>In the microcentrifuge tube containing fusogenic lipid-coated pSiNPs, add 100 &#181;L of the 1 mg/mL peptide stock and pipet gently. Keep the tube static at room temperature for 20 min (alternatively, &#62; 2 h at 4 &#176;C).</li>
		<li>To remove excess peptide or siRNA and other excipients, wash in a 30 kDa centrifugal filter by spinning at 5,000 x g at 25 &#176;C for 1 h. Centrifuge twice more with 1 mL of PBS under the same setting.</li>
		<li>Resuspend the final peptide-conjugated fusogenic lipid-coated pSiNPs in PBS at the desired concentration. The particles can now be aliquoted and frozen at -80 &#176;C for storage of at least 30 days.</li>
	</ol>
	</li>
</ol>

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MJS is a scientific founder of Spinnaker Biosciences, Inc., and has an equity interest in the company.&#160; Although this grant has been identified for conflict of interest management based on the overall scope of the project and its potential benefit to Spinnaker Biosciences, Inc., the research findings included in this particular publication may not necessarily relate to the interests of Spinnaker Biosciences, Inc. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. Other authors have nothing to disclose.

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.

<table><tbody><tr><td>1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)</td><td>Avanti Polar Lipids</td><td>850345P</td><td>Powder</td></tr><tr><td>1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP)</td><td>Avanti Polar Lipids</td><td>890890P</td><td>Powder</td></tr><tr><td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG(2000) Maleimide)</td><td>Avanti Polar Lipids</td><td>880126P</td><td>Powder</td></tr><tr><td>Aluminum foil</td><td>VWR International, LLC</td><td>12175-001</td><td></td></tr><tr><td>Calcium chloride (CaCl2)</td><td>Spectrum</td><td>C1977</td><td>Anhydrous, Pellets</td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>C6061</td><td></td></tr><tr><td>Computer</td><td>Dell</td><td>Dimension 9200</td><td>Any computer with PCI card slot is acceptable</td></tr><tr><td>Dil Stain (1,1&#039;-Dioctadecyl-3,3,3&#039;,3&#039;-Tetramethylindocarbocyanine Perchlorate (&#039;DiI&#039;; DiIC18(3)))</td><td>Life Technologies</td><td>D3911</td><td></td></tr><tr><td>Ethanol (EtOH)</td><td>UCSD Store</td><td>111</td><td>200 Proof</td></tr><tr><td>Hydrofluric acid (HF)</td><td>VWR International, LLC</td><td>MK264008&amp;nbsp;</td><td>Purity: 48%</td></tr><tr><td>Keithley 2651a Sourcemeter</td><td>Keithley</td><td>2651A</td><td></td></tr><tr><td>LabVIEW</td><td>National Instruments</td><td></td><td>Sample program available at: http://sailorgroup.ucsd.edu/sofware/</td></tr><tr><td>LysoTracker Green DND-26</td><td>Thermo Fisher Scientific</td><td>L7526</td><td></td></tr><tr><td>Liposome extrusion set with heating block</td><td>Avanti Polar Lipids</td><td>610000</td><td></td></tr><tr><td>Microcon-30kDa Centrifugal Filter Unit</td><td>EMD Millipore</td><td>MRCF0R030</td><td></td></tr><tr><td>O-ring</td><td>ChemGlass</td><td>CG-305-220</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Thermo Fisher Scientific</td><td>10010-049</td><td></td></tr><tr><td>Platinum coil</td><td>VWR International, LLC</td><td>AA10285-BU</td><td></td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Fisher Scientific</td><td>P250-3</td><td></td></tr><tr><td>Silicon wafer</td><td>Siltronix</td><td>Custom order</td><td></td></tr><tr><td>siRNA</td><td>Dharmacon</td><td>Custom order</td><td>IRF5, sense 5&amp;rsquo;-dTdT-CUG CAG AGA AUA ACC CUG A-dTdT-3&amp;rsquo; and antisense 5&amp;rsquo;-dTdT UCA GGG UUA UUC UCU GCA G dTdT-3&amp;rsquo;</td></tr><tr><td>Sonicator</td><td>VWR International, LLC</td><td>97043-960</td><td></td></tr><tr><td>Targeting peptide (CRV)</td><td>CPC Scientific</td><td>Custom order</td><td>sequence CRVLRSGSC; made cyclic by a disulfide bond between the side chains of the two cysteine residues</td></tr><tr><td>Teflon etch cell</td><td>Interface Performance Materials, Inc.</td><td>Custom order</td><td></td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>10977015</td><td></td></tr></tbody></table>

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 ...

synthesis-functionalization-characterization-fusogenic-porous-silicon

Universidad San Sebastian

Research

JoVE Journal

Engineering

7.5K Views.  University of California, San Diego. 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.

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

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Chemistry

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Bioengineering

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

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.

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.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based ...

Biochemistry

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...