The authors acknowledge financial support from Houston Methodist Research Institute, the National Natural Science Foundation of China (Nos., 21231007 and 21121061), the Ministry of Education of China (Nos., 20100171110013 and 313058), the National Basic Research Program of China (973 Program No. 2014CB845604), and the Fundamental Research Funds for the Central Universities.

1. PCPS Particle Preparation
<ol>
	<li>Oxidize non-functionalized porous silicon particles in a 30% solution of hydrogen peroxide at 95 &#176;C for 2 hr. Aminate the oxidized particles in 2% (3-&#8203;aminopropyl)&#8203;triethoxysilane solution in isopropyl alcohol for 2 days at 65 &#176;C with gentle stirring.</li>
	<li>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.</li>
	<li>Add a known volume of particle suspension (e.g., 10 &#956;l) to 10 ml isotone diluent and count the particles with a particle counting analyzer to determine the concentration of particles in the stock solution.</li>
	<li>Activate the acid group of L-arginine (0.1 nmol) with N-(3-dimethylaminopropyl)-N&#8242;-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.</li>
	<li>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.</li>
	<li>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 &#176;C with gentle stirring.</li>
	<li>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.</li>
	<li>Activate the second aspartic acid group of the Boc-Asp-OH/PEI solution with EDC (0.1 nmol)/NHS (0.1 nmol) at 4 &#176;C for 6 hr with gentle stirring.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. PCPS Particle Characterization
<ol>
	<li>Measure the size of the particles using a scanning electron microscope (SEM).
	<ol>
		<li>Place a drop of particle suspension (10,000 particles/&#956;l in ethanol) on a clean silica SEM sample stub and let dry at RT under vacuum.</li>
		<li>Measure SEM images at 8 kV with a 3&#8211;5 mm working distance using an in-lens detector.</li>
	</ol>
	</li>
	<li>Measure the zeta potential of the particles using a particle analyzer system.
	<ol>
		<li>Mix 10 &#956;l of particle suspension (10,000 particles/&#956;l in ethanol) with 1 ml of 10 mM phosphate buffer (pH 7.4).</li>
		<li>Load the sample into folded capillary cells and measure the zeta potential according to the manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
</ol>
3. Loading of siRNA into PCPS Particles
<ol>
	<li>Dry the PCPS particles (from PCPS particle preparation step 1.9) under vacuum O/N.</li>
	<li>Add siRNA (4 &#956;g) in nuclease-free water (20 &#956;l) to the dried PCPS particles and sonicate briefly. Use the following particle to siRNA ratios: 2 &#215; 105 particles/0.2 &#956;g siRNA, 4 &#215; 105 particles/0.2 &#956;g siRNA, 6 &#215; 105 particles/0.2 &#956;g siRNA, 8 &#215; 105 particles/0.2 &#956;g siRNA, 10 &#215; 105 particles/0.2 &#956;g siRNA and 12 &#215; 105 particles/0.2 &#956;g siRNA.</li>
	<li>Incubate for 3 hr at 4 &#176;C on a shaker (1,000 rpm) to allow siRNA binding to the particles.</li>
</ol>
4. Optimization of siRNA/PCPS Particle Ratio
<ol>
	<li>Add DNA loading dye to 20 &#956;l of the PCPS/control siRNA particles with different particle to siRNA ratios (see loading of siRNA into PCPS particles).</li>
	<li>Load the samples into a 2% agarose gel containing DNA gel stain.</li>
	<li>Perform electrophoresis at a constant voltage of 120 V for 20 min in running buffer.</li>
	<li>Analyze the gel with image acquisition and analysis software.</li>
</ol>
5. Release of siRNA from PCPS Particles
<ol>
	<li>Mix 20 &#956;l 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.</li>
	<li>Add DNA loading dye to the samples.</li>
	<li>Load samples into a 2% agarose gel containing DNA gel stain.</li>
	<li>Perform electrophoresis at a constant voltage of 120 V for 20 min in running buffer using DNA electrophoresis equipment and a power supply.</li>
	<li>Analyze the gel with image acquisition and analysis software.</li>
</ol>
6. Confocal Microscopy of PCPS Particles
<ol>
	<li>Add 5 &#956;l of PCPS/fluorescent control siRNA particles (10 &#215; 105 particles/0.2 &#956;g siRNA/20 &#956;l) to a glass cover slip.</li>
	<li>Visualize particle layers by confocal microscopy.</li>
</ol>
7. Characterization of Arg-PEI/control siRNA Nanoparticles
<ol>
	<li>To degrade the silicon material and form Arg-PEI/control siRNA nanoparticles, add PCPS/siRNA particles (10 &#215; 106 PCPS particles/2 &#956;g siRNA) to 100 &#956;l of phosphate buffered saline and shake (1,000 rpm) at 37 &#176;C for 2 days.</li>
	<li>Centrifuge the sample for 30 min at 18,800 x g and collect the supernatant.</li>
	<li>Measure the size of the formed Arg-PEI/siRNA nanoparticles with dynamic light scattering (DLS).
	<ol>
		<li>Mix 10 &#956;l of the supernatant with 1 ml 10 mM phosphate buffer (pH 7.4) and place into a plastic cuvette.</li>
		<li>Measure the size of the particles using a particle analyzer system according to the manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
	<li>Determine the size and morphology of the formed Arg/PEI-siRNA nanoparticles with atomic force microscopy.
	<ol>
		<li>Take 10 &#181;l of the supernatant and place on a silicon wafer.</li>
		<li>Visualize the particles with atomic force microscopy (AFM).</li>
	</ol>
	</li>
</ol>
8. Cell Culture
<ol>
	<li>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 &#176;C.</li>
</ol>
9. Confocal Microscopy of Live Cells with PCPS Particles
<ol>
	<li>Plate cells in 2-well culture slides at a seeding density of 3 x 105 cells/well for 24 hr.</li>
	<li>Add PCPS particles loaded with fluorescent control siRNA (10 &#215; 105 particles/0.2 &#956;g particle to siRNA ratio, 50 nM siRNA) to cells.</li>
	<li>Record a movie of the cells with a confocal microscope (supplied with a chamber, 5% CO2, 95% humidity and 37 &#176;C) for 12 hr following particle exposure.</li>
</ol>
10. Confocal Microscopy of Fixed Cells with PCPS Particles
<ol>
	<li>Plate cells in 2-well culture slides at a seeding density of 3 x 105 cells/well for 24 hr.</li>
	<li>Add PCPS particles loaded with fluorescent control siRNA (10 &#215; 105 particles/0.2 &#956;g particle to siRNA ratio, 50 nM siRNA) to cells and incubate for 1 day, 7 days and 10 days.</li>
	<li>Wash the cells twice with phosphate buffered saline and then fix them with 4% paraformaldehyde solution for 10 min.</li>
	<li>Wash the cells with phosphate buffered saline.</li>
	<li>Permeabilize the cells with 0.1% octyl phenol ethoxylate for 10 min and then wash them three times with phosphate buffered saline.</li>
	<li>Block the cells with albumin from bovine serum (10 mg/ml) in phosphate buffered saline for 10 min at RT with gentle stirring.</li>
	<li>To visualize filamentous actin, incubate the cells with fluorescently labeled phalloidin (1 &#956;l/40 &#956;l blocking solution) for 20 min at RT with gentle stirring and then wash in phosphate buffered saline.</li>
	<li>Remove the slides from the frame and add antifade reagent with 4&#39;,6-diamidino-2-phenylindole (DAPI) to visualize the nucleus.</li>
	<li>Add a cover glass on top and take images of the cells with confocal microscopy.</li>
</ol>
11. Flow Cytometry of Cells with PCPS/fluorescent Control siRNA Particles
<ol>
	<li>Plate cells in a 6-well plate at a seeding density of 3 x 105 cells/well for 24 hr.</li>
	<li>Add PCPS particles loaded with fluorescent control siRNA (10 &#215; 105 particles/0.2 &#956;g particle to siRNA ratio, 50 nM siRNA) to the cells and incubate for 24 hr.</li>
	<li>Wash the cells with phosphate buffered saline and scrape them with a cell scraper.</li>
	<li>Store the cells in phosphate buffered saline with 2% fetal bovine serum prior to analysis. Use untreated cells as a negative control.</li>
	<li>Perform flow cytometry.</li>
</ol>
12. Cell Viability of Cells with PCPS Particles and PCPS/control siRNA Particles
<ol>
	<li>Plate cells in a 96-well plate at a cell density of 3 &#215; 103 cells/well for 24 hr.</li>
	<li>Treat the cells with PCPS particles (1.5 &#215; 105/well and 6 &#215; 105/well) or PCPS/control siRNA particles (10 &#215; 105 particles/0.2 &#956;g 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.</li>
	<li>Perform a cell proliferation assay according to the manufacturer&#8217;s instructions.</li>
	<li>Represent data as the mean &#177; standard deviation.</li>
</ol>
13. Western Blot of Cells with PCPS/ATM Mutated siRNA Particles
<ol>
	<li>Plate cells in a 6-well plate at a cell density 2 &#215; 105 cells/well for 24 hr.</li>
	<li>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.</li>
	<li>Lyse the cells using a protein extraction reagent supplemented with a protease inhibitor cocktail.</li>
	<li>Centrifuge the cell lysates for 10 min at 14,000 x g and recover the supernatant.</li>
	<li>Determine the protein concentration with a protein quantification assay according to the manufacturer&#8217;s instructions.</li>
	<li>Add sample loading buffer (with 5 &#956;l 2-mercaptoethanol/ml buffer) to the samples and heat them for 6 min at 99 &#176;C.</li>
	<li>Load the protein samples (20 &#956;g/&#956;l) 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.</li>
	<li>Transfer the gel in transfer buffer (with 20% methanol) to a nitrocellulose membrane (1 hr, 100 V) using electrophoresis equipment and a power supply.</li>
	<li>Block the membrane with 5% dry milk for 1 hr.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>For the loading control, wash the membrane and repeat steps 13.9-13.12 using a &#946;-actin primary antibody (from mouse, 1:10,000 dilution) and a secondary antibody (anti-mouse 1:4,000 dilution).</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+delivery+systems+for+cancer+treatment.">siRNA delivery systems for cancer treatment.</a> Adv Drug Deliv Rev. 61 (10), 850-862 (2009).</li><li>Mintzer, M. A., Simanek, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonviral+vectors+for+gene+delivery.">Nonviral vectors for gene delivery.</a> Chem Rev. 109 (2), 259-302 (2009).</li><li>Whitehead, K. A., Langer, R., Anderson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knocking+down+barriers:+advances+in+siRNA+delivery.">Knocking down barriers: advances in siRNA delivery.</a> Nat Rev Drug Discov. 8 (2), 129-138 (2009).</li><li>Decuzzi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+shape+effects+in+the+biodistribution+of+intravascularly+injected+particles.">Size and shape effects in the biodistribution of intravascularly injected particles.</a> J Control Release. 141 (3), 320-327 (2010).</li><li>Decuzzi, P., Ferrari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+maps+for+nanoparticles+targeting+the+diseased+microvasculature.">Design maps for nanoparticles targeting the diseased microvasculature.</a> Biomaterials. 29 (3), 377-384 (2008).</li><li>Martinez, J. 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The authors declare that they have no competing financial interests.

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...

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 applications1-5. 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 agents6,7. 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 model9, 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 degrades11, 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.

<table><tbody><tr><td>Polyethylenimine (PEI), branched</td><td>Sigma-Aldrich</td><td>408727</td><td>Average molecular weight ~25,000&amp;nbsp;Da</td></tr><tr><td>L-Arginine</td><td>Sigma-Aldrich</td><td>A5006</td><td>Reagent grade, &amp;ge;98%</td></tr><tr><td>Boc-Asp-OH</td><td>Sigma-Aldrich</td><td>408-468</td><td>99%</td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich</td><td>216763</td><td>30&amp;nbsp;wt. % in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>339741</td><td>100.00%</td></tr><tr><td>Isopropyl alcohol</td><td>Sigma-Aldrich</td><td>W292907</td><td>&amp;ge;99.7%</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>459844</td><td>&amp;ge;99.5%&amp;nbsp;</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-Dimethylaminopropyl)-&lt;em&gt;N&amp;prime;&lt;/em&gt;-ethylcarbodiimide hydrochloride (EDC)</td><td>Sigma-Aldrich</td><td>03449</td><td>&amp;ge;99%</td></tr><tr><td>Albumin from bovine serum</td><td>Sigma-Aldrich</td><td>A7030-10G</td><td>Blocking agent</td></tr><tr><td>Ataxia-telangiectasia mutated siRNA&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td>Designed in-house</td></tr><tr><td>Tris Acetate-EDTA buffer</td><td>Sigma-Aldrich</td><td>T9650</td><td>For DNA agarose gel electrophoresis</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma-Aldrich</td><td>P1379</td><td>Polyethylene glycol sorbitan monolaurate</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma Aldrich</td><td>M6250</td><td>For Western blot</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>71496</td><td>For making phosphate buffer&amp;nbsp;</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>71640</td><td>For making phosphate buffer</td></tr><tr><td>Anti-Mouse IgG</td><td>Sigma-Aldrich</td><td>A4416</td><td>Secondary antibody (anti-mouse) for Western blot</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Hydroxysuccinimide (NHS)</td><td>Sigma-Aldrich</td><td>130672</td><td>98%</td></tr><tr><td>CELLSTAR 96W Microplate Tissue Culture Treated Clear w/ Lid</td><td>Greiner Bio-One</td><td>655182</td><td>96-well plate</td></tr><tr><td>10x Tris-Glycine Liquid</td><td>Li-Cor&amp;nbsp;</td><td>928-40010</td><td>Transfer buffer for Western blot</td></tr><tr><td>Paraformaldehyde solution 4% in PBS&amp;nbsp;</td><td>Santa Cruz</td><td>Sc-281692</td><td>Fixation of cells</td></tr><tr><td>CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS)</td><td>Promega</td><td>G5421</td><td>Proliferation assay</td></tr><tr><td>Phosphate buffered saline</td><td>Fisher Scientific</td><td>BP399-500</td><td>10x Solution</td></tr><tr><td>Corning cellgro Dulbecco&#039;s Modification of Eagle&#039;s (Mod.)</td><td>Fisher Scientific&amp;nbsp;</td><td>MT-15-017-CM</td><td>Cell culture media, 1x solution</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>AC21568-2500</td><td>Octyl phenol ethoxylate, permeabilization agent</td></tr><tr><td>Cover glass</td><td>Fisher Scientific</td><td>12-530C&amp;nbsp;</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific&amp;nbsp;</td><td>A412-1</td><td>For Western blot transfer buffer</td></tr><tr><td>Plastic Cuvettes</td><td>Fisher Scientific&amp;nbsp;</td><td>14-377-010&amp;nbsp;</td><td>For size measurements using Zetasizer Nano ZS</td></tr><tr><td>Molecular BioProducts RNase AWAY Surface Decontaminant</td><td>Fisher Scientific</td><td>14-754-34</td><td>Spray for removing RNAse contamination</td></tr><tr><td>Agarose</td><td>Fisher Scientific</td><td>BP165-25</td><td>Low melting point, for running RNA samples</td></tr><tr><td>ProLong Gold Antifade Reagent with DAPI&amp;nbsp;</td><td>Invitrogen</td><td>P36935</td><td>Antifade reagent with DAPI, nucelus detection</td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Invitrogen</td><td>A12379</td><td>Dissolve 300 units in 1.5&amp;nbsp;ml methanol, detection of filamentous actin</td></tr><tr><td>SYBR Safe DNA Gel Stain</td><td>Invitrogen</td><td>S33102</td><td>Visualization of RNA</td></tr><tr><td>Negative Control siRNA</td><td>Qiagen</td><td>1022076</td><td>Control siRNA</td></tr><tr><td>AllStars Neg. siRNA AF 555</td><td>Qiagen</td><td>1027294</td><td>Fluorescent control siRNA</td></tr><tr><td>Cell scraper</td><td>Celltreat</td><td>229310</td><td></td></tr><tr><td>BioLite Multidishes and Microwell Plates</td><td>Thermo Scientific</td><td>130184</td><td>6-well plate</td></tr><tr><td>Pierce LDS Sample Loading Buffer (4x)</td><td>Thermo Scientific</td><td>84788</td><td>Sample loading buffer for Western blot</td></tr><tr><td>Pierce BCS Protein Assay Kit</td><td>Thermo Scientific</td><td>23227</td><td>Protein quantification assay</td></tr><tr><td>Halt Protease Inhibitor Single-Use Cocktails (100x)</td><td>Thermo Scientific</td><td>78430</td><td>Protease inhibitor cocktail, use at 1x</td></tr><tr><td>M-PER Mammalian Protein Extraction Reagent</td><td>Thermo Scientific&amp;nbsp;</td><td>78501</td><td>Protein extraction reagent</td></tr><tr><td>Sorvall Legend Micro 21R</td><td>Thermo Scientific</td><td>75002440</td><td>Centrifuge</td></tr><tr><td>Beta Actin Antibody&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>MA1-91399</td><td>&amp;beta;-actin primary antibody (from mouse) for western blot</td></tr><tr><td>6x TriTrack DNA Loading Dye</td><td>Thermo Scientific</td><td>R1161</td><td>DNA loading dye</td></tr><tr><td>Nuclease-Free Water&amp;nbsp;</td><td>Life Technologies</td><td>AM9938</td><td></td></tr><tr><td>Non-Fat Dry Milk</td><td>Lab Scientific</td><td>M0841</td><td>For Western blot</td></tr><tr><td>2-well BD Falcon culture slides&amp;nbsp;</td><td>BD Biosciences</td><td>354102</td><td>2-well culture slides</td></tr><tr><td>Amersham ECL Western blot detection reagent</td><td>GE Healthcare Life Sciences</td><td>RPN2106</td><td>Western blot detection reagent</td></tr><tr><td>BA Membranes</td><td>GE Healthcare Life Sciences</td><td>10402096</td><td>Nitrocellulose membrane for Wester blot</td></tr><tr><td>ATM (D2E2) Rabbit mAb</td><td>Cell Signaling</td><td>2873S</td><td>ATM primary antibody (from rabbit) for Western blot</td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell Signaling&amp;nbsp;</td><td>7074</td><td>Secondary antibody (anti-rabbit) for Western blot&amp;nbsp;</td></tr><tr><td>Folded capillary cells</td><td>Malvern&amp;nbsp;</td><td>DTS 1061</td><td>For zeta potentail measurements using Zetasizer Nano ZS</td></tr><tr><td>MDA-MB-231 cell line</td><td>ATCC</td><td>HTB-26</td><td>Mammary Gland/Breast</td></tr><tr><td>12% Mini-PROTEAN TGX Gel</td><td>Bio-rad</td><td>456-1043&amp;nbsp;</td><td>For Western blot</td></tr><tr><td>Biorad PowerPac HC</td><td>Bio-rad</td><td>164-5052</td><td>Power supply for electrophoresis</td></tr><tr><td>10x Tris/Glycine/SDS Buffer</td><td>Bio-rad</td><td>161-0732</td><td>Running buffer for Western blot</td></tr><tr><td>Wide Mini-Sub Cell GT Cell</td><td>Bio-rad</td><td>170-4405</td><td>Electrophoresis equipment for DNA agarose gel&amp;nbsp;</td></tr><tr><td>Mini-PROTEAN Tetra cell</td><td>Bio-rad</td><td>165-8000</td><td>Electrophoresis equipment for Western blot</td></tr><tr><td>ChemiDoc XRS+ System with Image Lab Software</td><td>Bio-rad</td><td>170-8265</td><td>Image acquisition and analysis software for gels and blots</td></tr><tr><td>4&quot; (10&amp;nbsp;cm) dia., 5&amp;nbsp;x&amp;nbsp;7&amp;nbsp;mm diced Silicon Wafer</td><td>Ted Pella&amp;nbsp;</td><td>16007</td><td>Silicon waferfor scanning electron microscopy and atomic force microscopy</td></tr><tr><td>Thermomixer R</td><td>Eppendorf</td><td>22670107</td><td>Shaker&amp;nbsp;</td></tr><tr><td>Isoton II diluent</td><td>Beckman Coulter</td><td>8546719</td><td>Isoton diluent</td></tr><tr><td>Multisizer 4 Coulter Counter</td><td>Beckman Coulter</td><td>A63076</td><td>Particle counting analyzer</td></tr><tr><td>Non-functionalized porous silicon particles&amp;nbsp;</td><td>Microelectronics Research Center, University of Texas at Austin</td><td></td><td>Dicoidal shape. 2.6 &amp;mu;m (diameter) x 0.7 &amp;mu;m (height), provided in isopropyl alcohol</td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern</td><td></td><td>Particle analyzer system for size and zeta potential&amp;nbsp;</td></tr><tr><td>Scanning Electron Microscope</td><td>FEI</td><td></td><td>Particle size and shape&amp;nbsp;</td></tr><tr><td>Atomic Force Microscope</td><td>Bruker</td><td></td><td>Particle size and shape</td></tr><tr><td>Fluo ViewTM 1000 Confocal Microscope</td><td>Olympus</td><td></td><td>Visualization of fixed and live cells</td></tr></tbody></table>

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.

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 &#956;m (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 ...

Watch this Scientific Journal Video about Porous Silicon Microparticles for Delivery of siRNA Therapeutics at JoVE.com

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

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 ...

porous-silicon-microparticles-for-delivery-of-sirna-therapeutics

Research

JoVE Journal

Bioengineering

10.9K Views.  Houston Methodist Research Institute.  Levering blijft de belangrijkste uitdaging voor de therapeutische toepassing van kleine interfererende RNA (siRNA). Dit protocol omvat het gebruik van een multifunctionele en biocompatibele siRNA afleverplateau, bestaande uit arginine en polyethyleenimine geënte poreuze silicium microdeeltjes. 

Video: Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Cancer Research

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Developmental Biology

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.

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 ...

Engineering

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  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 ...

Chemistry

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 ...

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 ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

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