The authors thank the National Institute of General Medical Sciences of the National Institutes of Health (NIH) for financial support through an Institutional Development Award (IDeA) under grant number P20GM103541 as well as grant number P20GM10344615. The statements herein do not reflect the views of the NIH. We also acknowledge the Delaware Biotechnology Institute (DBI) and Delaware Economic Development Office (DEDO) for financial support through the Bioscience Center for Advanced Technology (Bioscience CAT) award (12A00448).

1. Formulation of siRNA Nanocarriers
<ol>
	<li>Prepare separate solutions of siRNA and mPEG-b-P(APNBMA) with equal volumes diluted in 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer at pH 6.0.
	<ol>
		<li>Add siRNA at a concentration of 32 &#181;g/mL to 20 mM HEPES solution. 
		NOTE: The siRNA was a non-targeted, universal negative control sequence; however, the siRNA can be designed to target any gene of interest.</li>
		<li>Dissolve mPEG-b-P(APNBMA) polymers into a 20 mM HEPES solution. Add an appropriate amount of mPEG-b-P(APNBMA) to make a 220 &#181;g/mL solution so that the N/P ratio (N, amine groups on mPEG-b-P(APNBMA); P, phosphate groups on siRNA) is 4. 
		NOTE: The synthetic protocol for the mPEG-b-P(APNBMA) polymers is reported elsewhere30.</li>
	</ol>
	</li>
	<li>Add the mPEG-b-P(APNBMA) solution dropwise to an equal volume of the siRNA solution while gently mixing on a vortex machine. Continue to vortex for 30 s following polymer addition. Incubate the samples in the dark at room temperature for 30 min.</li>
</ol>
2. Measuring siRNA Release Using Gel Electrophoresis
<ol>
	<li>Formulate the nanocarriers according to steps 1.1-1.2, and scale the volumes to accommodate the number of samples desired.</li>
	<li>Mix the nanocarrier with sodium dodecyl sulfate (SDS).
	<ol>
		<li>Prepare a 1 mg/mL solution of SDS in water. Aliquot out the amount of SDS solution needed to produce solutions with an S/P ratio (S, sulfate groups on SDS; P, phosphate groups on siRNA) of 15. 
		NOTE: If the polyplex solution contains 1 &#181;g of siRNA, 13 &#181;g of SDS must be added to achieve an S/P ratio of 15.</li>
		<li>Add the SDS solution to each nanocarrier solution dropwise while gently mixing on a vortex machine. Continue to vortex for 30 s following SDS addition.</li>
		<li>Centrifuge the samples at 3,000 x g for 5 s. Incubate the samples in the dark at room temperature for 30 min.</li>
	</ol>
	</li>
	<li>Calibrate and set a UV laser with a 365 nm filter to an intensity of 200 W/m. Ensure that the light intensity is measured from the location at which the bottom of the sample solution will be seated.</li>
	<li>Load the nanocarrier/SDS solution into a glass chamber comprised of glass slides separated by a rubber gasket.
	<ol>
		<li>Pre-wash glass slides in a 7:3 (v/v) ethanol/water solution in water and dry completely. Cut a hole (~2 x 3 cm rectangle) into a rubber gasket. Place the rubber gasket onto a glass slide.</li>
		<li>Pipette the nanocarrier/SDS solution onto the glass slide inside the hole of the rubber gasket. Load an excess of solution (20 &#181;L in surplus) onto the glass slide while avoiding contact with the rubber gasket. 
		NOTE: Some liquid will be lost during the subsequent steps.</li>
		<li>Place the second glass slide on top of the slide-gasket. To avoid air bubble generation, place one end of the slide down first and then slowly lower the other end.</li>
		<li>Attach binder clips to each side of the glass chamber to hold it closed.</li>
	</ol>
	</li>
	<li>Irradiate the samples for the desired length of time (e.g., 0-60 min) using the UV laser with a 365 nm filter at an intensity of 200 W/m. Remove the binder clips and open the chamber.</li>
	<li>Pipette 25 &#181;L of the irradiated nanocarrier/SDS samples into a microcentrifuge tube. Incubate the solutions in the dark at room temperature for 30 min.</li>
	<li>Prepare a 2 wt% agarose gel pre-stained with 0.5 &#181;g/mL ethidium bromide in Tris/Borate/EDTA (TBE) buffered solution according to standard protocols31. Prepare a loading buffer comprised of 3:7 (v/v) glycerol/water.</li>
	<li>Add 5 &#181;L of the loading buffer solution to 25 &#181;L of each nanocarrier/SDS sample. Incubate the samples in the dark at room temperature for 10 min.</li>
	<li>Load 30 &#181;L of each nanocarrier/SDS sample into the 2% agarose gel. Run the gel in the dark at 100 V for 30 min. Image the gel using a gel imaging system with ethidium bromide filters. Save the gel image files and proceed to step 2.10 for band intensity quantification. Ensure that the band intensities are bright enough to clearly visualize but not too bright that the signals are saturated.</li>
	<li>Quantify the band intensities using publicly available ImageJ software32.
	<ol>
		<li>Using the ROI tool of ImageJ, determine the fluorescence intensity of the free siRNA bands in each lane by drawing a rectangle around each band. Plot the intensity curves of each lane, and integrate the area under the curves by drawing a horizontal line across the intensity curves and clicking the tracing wand inside the enclosed areas.</li>
		<li>Compute the relative intensity of each lane by dividing the area under the curve of each sample by the area under the curve of the siRNA positive control (no mPEG-b-P(APNBMA) added and no SDS added). Report the percentage of siRNA released as the normalized band intensity of each sample.</li>
	</ol>
	</li>
</ol>
3. Measuring siRNA Release Using Fluorescence Correlation Spectroscopy (FCS)
<ol>
	<li>Obtain siRNA labelled with a single fluorophore at the 5&#39; end of the sense strand. 
	NOTE: The siRNA can be purchased pre-annealed with the labels conjugated in the desired location. The fluorophore should be photo-stable and absorb/emit between 450 and 750 nm to avoid UV light quenching and energy transfer with mPEG-b-P(APNBMA).</li>
	<li>Formulate the nanocarriers according to steps 1.1-1.2 using the labelled siRNA. Scale the volumes to accommodate the number of samples desired.</li>
	<li>Incubate the solutions in SDS and irradiate for the desired length of time according to steps 2.2-2.6.</li>
	<li>Preparation of FCS sample chamber.
	<ol>
		<li>Wash a glass slide with a 7:3 (v/v) ethanol/water solution and completely dry the glass using a wipe and an air stream.</li>
		<li>Remove the pieces of paper from a double-sided adhesive spacer to expose the double-sided adhesive spacer. Attach the spacer to a glass coverslip.</li>
		<li>Pipette the nanocarrier/SDS solution onto the cover slip in the middle of the hole from the adhesive spacer.</li>
		<li>Place the glass slide on top of the coverslip. Push on the glass slide to ensure that the glass slide and coverslip are well attached and form a seal.</li>
	</ol>
	</li>
	<li>Use a confocal microscope for FCS measurements33. Use a 40X water immersion apochromat objective with a numerical aperture of 1.2. Use the appropriate excitation laser channel (488 nm) to collect at least 30 measurements of 10 s each per sample34. Ensure that the laser intensity and detector alignment remain the same for each sample.
	<ol>
		<li>In addition to the experimental samples, measure controls including: a blank sample without labelled siRNA; and a free siRNA sample with labelled siRNA but no mPEG-b-P(APNBMA).</li>
	</ol>
	</li>
	<li>Analyze the data using FCS-specific software. Identify the baseline count rate of each sample by determining the stable count rate during a time when no nanocarriers are passing through the confocal volume29.
	<ol>
		<li>Subtract the count rate of the blank sample from every sample baseline count rate value. Normalize the resulting values to the free siRNA control to calculate the percent of free siRNA35.</li>
	</ol>
	</li>
</ol>
4. Kinetic Modeling to Predict Gene Silencing
<ol>
	<li>Create scripts in a mathematical programming language using the simple set of ordinary differential equations to predict gene silencing29. 
	NOTE: Scripts can be made available upon request.
	<ol>
		<li>Write the set of ordinary differential equations as: 
		<img alt="Equation 1" src="/files/ftp_upload/55803/55803eq1.jpg" />&#160;&#160;&#160;&#160; (1) 
		<img alt="Equation 2" src="/files/ftp_upload/55803/55803eq2.jpg" />&#160;&#160;&#160;&#160; (2) 
		<img alt="Equation 3" src="/files/ftp_upload/55803/55803eq3.jpg" />&#160;&#160;&#160;&#160; (3) 
		NOTE: For equations 1&#8722;3, the terms kmRNA, ksiRNA, and kprot are the rate constants for the production of mRNA, siRNA, and protein, respectively. The terms km,deg, ks,deg, and kp,deg are the rate constants for the degradation of mRNA, siRNA, and protein, respectively. Degradation rate constants are computed on the basis of the component half-lives, and production rate constants are fit to ensure that mRNA and protein steady-state values are reached in the absence of siRNA.
		<ol>
			<li>Determine the half-lives of the mRNA and protein for the gene(s) of interest, either experimentally as described in reference 36 or from the literature (see the Discussion). Also, determine the doubling time for the cell line. Input these values into the appropriate degradation rate expressions.</li>
			<li>Tune the production rate constants so that gene expression levels remain steady at a normalized value of 100 if no siRNA is introduced. Specifically, set [siRNA] to zero and vary the values of the kmRNA, ksiRNA, and kprot production rate constants until [mRNA] and [protein] remain within 1% of the initial normalized value of 100% for the duration of the simulation.</li>
			<li>Using the relative amounts of siRNA released from the previously described gel electrophoresis and FCS assays as estimates, adjust the initial relative concentration of siRNA in the script. Specifically, vary [siRNA] to be proportional to the relative amount of released siRNA, with a value of 100 corresponding to the maximum amount29.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Cell Culture and In Vitro siRNA Delivery
<ol>
	<li>Culture NIH/3T3 murine embryonic fibroblasts according to the protocols from the supplier.
	<ol>
		<li>Grow the cells in growth medium (Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin). Maintain the cells at 37 &#176;C in a humidified environment with 5% CO2.</li>
	</ol>
	</li>
	<li>Seed the cells in 6-well tissue culture treated plates.
	<ol>
		<li>Follow the recommended subculturing procedure from the supplier. Count the cells using a hemocytometer. Dilute the cells in supplemented growth media to a concentration of 75,000 cells/mL.</li>
		<li>Add 2 mL of cell suspension (75,000 cells/mL) to each well of the 6-well plate. Let the cells adhere and recover for 24 h in the incubator.</li>
	</ol>
	</li>
	<li>Prepare the cells for transfection by washing with phosphate-buffered saline (PBS) and adding 1.5 mL of serum- and antibiotic-free transfection medium (see the Table of Materials) to each well.</li>
	<li>Formulate the siRNA nanocarriers according to steps 1.1-1.2. Add 25 &#181;L of nanocarrier solution containing 30 pmol of siRNA to each well. Gently pipette the media up and down to mix. Place the cells in the incubator for 3 h.</li>
	<li>Remove the transfection media and wash each well with PBS. Add 1 mL of supplemented growth media and place the cells in the incubator to recover for 30 min.</li>
	<li>To prepare the cells for treatment with a photo-stimulus, remove the supplemented growth media. Add 1 mL of transfection media (without phenol red) to each well. 
	NOTE: Ensure that the transfection media does not contain phenol red.</li>
	<li>Calibrate and set a UV laser with a 365 nm filter to an intensity of 200 W/m. Ensure that the light intensity is measured from the location at which the bottom of the cell plate will be seated.</li>
	<li>Place the cells on a hot plate set to 37 &#176;C. Remove the plate cover of the cells. Irradiate the cells from above the plate for the desired time (up to 20 min) using the UV laser with a 365 nm filter at an intensity of 200 W m-2.</li>
	<li>Remove the transfection media and add 2 mL of supplemented growth media. Place in incubator until further analysis (e.g., 24 h for qPCR and 48 h for Western blotting).
	<ol>
		<li>Measure changes in gene expression using a variety of techniques such as Western blotting37 and qPCR.38 For genes with visible signals, such as GFP, use fluorescence microscopy29. 
		NOTE: These techniques are suggested due to their ease of use and accuracy in quantifying gene expression</li>
	</ol>
	</li>
</ol>
6. Controlling Gene Silencing in a Spatiotemporal Manner
<ol>
	<li>Culture, seed, and transfect cells according to steps 5.1-5.7.</li>
	<li>Prepare a photomask that completely blocks 365 nm light and minimizes reflections. 
	NOTE: In this case, 10 x 10 cm pieces of aluminum foil and black construction paper were used to block the light and to reduce reflections, respectively. The aluminum foil and construction paper were glued together to form a single unit.
	<ol>
		<li>Cut/punch/machine the desired shape into the photomask. For example, use a sharp-edged blade and a hole-puncher to form a straight-line pattern (~5 cm long) and a circular pattern (~7 mm diameter) in the photomask, respectively.</li>
	</ol>
	</li>
	<li>Glue the photomask to the bottom of the 6-well plate with the pattern centered under the well containing the cells with the anti-reflective side (e.g., black construction paper) facing the plate. Ensure that the glue is not placed near the edge (within ~3 mm) of the pattern.</li>
	<li>Set up two ring stands approximately 25 cm apart and attach a platform to each ring stand so that the platforms are of equal height. Suspend the cell plate between the two stands by resting the plate on top of the platforms. Ensure that the plate is level.</li>
	<li>Irradiate the cells from below the sample for the desired time (up to 20 min) using the UV laser with a 365 nm filter at an intensity of 200 W/m2.</li>
	<li>Remove the transfection media and add 2 mL of supplemented growth media. Place in incubator to recover for at least 24 h. Image the cells using fluorescence microscopy as described29.</li>
</ol>

<ol>
	<li>Forbes, D. C., Peppas, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+delivery+of+small+RNA+and+DNA.">Oral delivery of small RNA and DNA.</a> J Control Release. 162 (2), 438-445 (2012).</li><li>Davis, M. E., 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=Evidence+of+RNAi+in+humans+from+systemically+administered+siRNA+via+targeted+nanoparticles.">Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles.</a> Nature. 464 (7291), 1067-1070 (2010).</li><li>Bouchie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Companies+in+footrace+to+deliver+RNAi.">Companies in footrace to deliver RNAi.</a> Nat Biotechnol. 30 (12), 1154-1157 (2012).</li><li>Burke, P. A., Pun, S. H., Reineke, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+Polymeric+Delivery+Systems+Amidst+a+Nucleic+Acid+Therapy+Renaissance.">Advancing Polymeric Delivery Systems Amidst a Nucleic Acid Therapy Renaissance.</a> ACS Macro Lett. 2 (10), 928-934 (2013).</li><li>Gooding, M., Browne, L. P., Quinteiro, F. M., Selwood, D. L. <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:+From+Lipids+to+Cell-penetrating+Peptides+and+Their+Mimics.">siRNA Delivery: From Lipids to Cell-penetrating Peptides and Their Mimics.</a> Chem Biol Drug Des. 80 (6), 787-809 (2012).</li><li>Motta-Mena, L. B., 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=An+optogenetic+gene+expression+system+with+rapid+activation+and+deactivation+kinetics.">An optogenetic gene expression system with rapid activation and deactivation kinetics.</a> Nat Chem Biol. 10 (3), 196-202 (2014).</li><li>Wang, X., Chen, X., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+gene+expression+by+a+light-switchable+transgene+system.">Spatiotemporal control of gene expression by a light-switchable transgene system.</a> Nat Methods. 9 (3), 266-269 (2012).</li><li>Ziauddin, J., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays+of+cells+expressing+defined+cDNAs.">Microarrays of cells expressing defined cDNAs.</a> Nature. 411 (6833), 107-110 (2001).</li><li>Saltzman, W. M., Olbricht, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+drug+delivery+into+tissue+engineering.">Building drug delivery into tissue engineering.</a> Nat Rev Drug Discov. 1 (3), 177-186 (2002).</li><li>Mura, S., Nicolas, J., Couvreur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+nanocarriers+for+drug+delivery.">Stimuli-responsive nanocarriers for drug delivery.</a> Nat Mater. 12 (11), 991-1003 (2013).</li><li>Shim, M. S., Kwon, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+polymers+and+nanomaterials+for+gene+delivery+and+imaging+applications.">Stimuli-responsive polymers and nanomaterials for gene delivery and imaging applications.</a> Adv Drug Delivery Rev. 64 (11), 1046-1058 (2012).</li><li>Kelley, E. G., Albert, J. N. L., Sullivan, M. O., Epps, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+copolymer+solution+and+surface+assemblies+for+biomedical+applications.">Stimuli-responsive copolymer solution and surface assemblies for biomedical applications.</a> Chem Soc Rev. 42 (17), 7057-7071 (2013).</li><li>Weber, W., Fussenegger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+biomedical+applications+of+synthetic+biology.">Emerging biomedical applications of synthetic biology.</a> Nat Rev Genet. 13 (1), 21-35 (2012).</li><li>Konermann, S., 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=Optical+control+of+mammalian+endogenous+transcription+and+epigenetic+states.">Optical control of mammalian endogenous transcription and epigenetic states.</a> Nature. 500 (7463), 472-476 (2013).</li><li>Shimizu-Sato, S., Huq, E., Tepperman, J. M., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-switchable+gene+promoter+system.">A light-switchable gene promoter system.</a> Nat Biotechnol. 20 (10), 1041-1044 (2002).</li><li>Huschka, R., 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=Gene+Silencing+by+Gold+Nanoshell-Mediated+Delivery+and+Laser-Triggered+Release+of+Antisense+Oligonucleotide+and+siRNA.">Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA.</a> ACS Nano. 6 (9), 7681-7691 (2012).</li><li>Li, H. -. J., Wang, H. -. X., Sun, C. -. Y., Du, J. -. Z., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-detachable+nanoparticles+based+on+a+light-responsive+amphiphile+for+enhanced+siRNA+delivery.">Shell-detachable nanoparticles based on a light-responsive amphiphile for enhanced siRNA delivery.</a> R Soc Chem Adv. 4 (4), 1961-1964 (2014).</li><li>Braun, G. B., 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=Laser-Activated+Gene+Silencing+via+Gold+Nanoshell-siRNA+Conjugates.">Laser-Activated Gene Silencing via Gold Nanoshell-siRNA Conjugates.</a> ACS Nano. 3 (7), 2007-2015 (2009).</li><li>Gilleron, J., 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=Image-based+analysis+of+lipid+nanoparticle-mediated+siRNA+delivery,+intracellular+trafficking+and+endosomal+escape.">Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape.</a> Nat Biotechnol. 31 (7), 638-646 (2013).</li><li>Wittrup, A., 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=Visualizing+lipid-formulated+siRNA+release+from+endosomes+and+target+gene+knockdown.">Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown.</a> Nat Biotechnol. 33 (8), 870-876 (2015).</li><li>Roth, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurements+and+rational+materials+design+for+intracellular+delivery+of+oligonucleotides.">Quantitative measurements and rational materials design for intracellular delivery of oligonucleotides.</a> Biotechnol Prog. 24 (1), 23-28 (2008).</li><li>Mao, S., 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=Influence+of+polyethylene+glycol+chain+length+on+the+physicochemical+and+biological+properties+of+poly(ethylene+imine)-graft-poly(ethylene+glycol)+block+copolymer/SiRNA+polyplexes.">Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes.</a> Bioconjugate Chem. 17 (5), 1209-1218 (2006).</li><li>Raab, R. M., Stephanopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+gene+silencing+by+RNA+interference.">Dynamics of gene silencing by RNA interference.</a> Biotechnol Bioeng. 88 (1), 121-132 (2004).</li><li>Cuccato, G., 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=Modeling+RNA+interference+in+mammalian+cells.">Modeling RNA interference in mammalian cells.</a> BMC Systems Biology. 5, 1 (2011).</li><li>Varga, C. M., Hong, K., Lauffenburger, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+synthetic+gene+delivery+vector+design+properties.">Quantitative analysis of synthetic gene delivery vector design properties.</a> Mol Ther. 4 (5), 438-446 (2001).</li><li>Chen, H. H., 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=Quantitative+comparison+of+intracellular+unpacking+kinetics+of+polyplexes+by+a+model+constructed+from+quantum+Dot-FRET.">Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum Dot-FRET.</a> Mol Ther. 16 (2), 324-332 (2008).</li><li>Bartlett, D. W., Davis, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+kinetics+of+siRNA-mediated+gene+silencing+from+live-cell+and+live-animal+bioluminescent+imaging.">Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging.</a> Nucleic Acids Res. 34 (1), 322-333 (2006).</li><li>Foster, A. A., Greco, C. T., Green, M. D., Epps, T. H., Sullivan, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Mediated+Activation+of+siRNA+Release+in+Diblock+Copolymer+Assemblies+for+Controlled+Gene+Silencing.">Light-Mediated Activation of siRNA Release in Diblock Copolymer Assemblies for Controlled Gene Silencing.</a> Adv Healthc Mater. 4 (5), 760-770 (2015).</li><li>Greco, C. T., Epps, T. H., Sullivan, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+Design+of+Polymer+Nanocarriers+to+Spatiotemporally+Control+Gene+Silencing.">Mechanistic Design of Polymer Nanocarriers to Spatiotemporally Control Gene Silencing.</a> ACS Biomater Sci Eng. 2 (9), 1582-1594 (2016).</li><li>Green, M. D., 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=Catch+and+release:+photocleavable+cationic+diblock+copolymers+as+a+potential+platform+for+nucleic+acid+delivery.">Catch and release: photocleavable cationic diblock copolymers as a potential platform for nucleic acid delivery.</a> Polym Chem. 5 (19), 5535-5541 (2014).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Marquer, C., Leveque-Fort, S., Potier, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Lipid+Raft+Partitioning+of+Fluorescently-tagged+Probes+in+Living+Cells+by+Fluorescence+Correlation+Spectroscopy+(FCS).">Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS).</a> J Vis Exp. (62), (2012).</li><li>Staaf, E., Bagawath-Singh, S., Johansson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Diffusion+in+Plasma+Membranes+of+Primary+Lymphocytes+Measured+by+Fluorescence+Correlation+Spectroscopy.">Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy.</a> J Vis Exp. (120), e54756 (2017).</li><li>Buyens, K., 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=Monitoring+the+disassembly+of+siRNA+polyplexes+in+serum+is+crucial+for+predicting+their+biological+efficacy.">Monitoring the disassembly of siRNA polyplexes in serum is crucial for predicting their biological efficacy.</a> J Control Release. 141 (1), 38-41 (2010).</li><li>Eden, E., 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=Proteome+Half-Life+Dynamics+in+Living+Human+Cells.">Proteome Half-Life Dynamics in Living Human Cells.</a> Science. 331 (6018), 764-768 (2011).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad. Sci U S A. 76 (9), 4350-4354 (1979).</li><li>Wittwer, C. T., Herrmann, M. G., Moss, A. A., Rasmussen, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Fluorescence+Monitoring+of+Rapid+Cycle+DNA+Amplification.">Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification.</a> Biotechniques. 54 (6), 314-320 (2013).</li><li>Cho, S. K., Dang, C., Wang, X., Ragan, R., Kwon, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixing-sequence-dependent+nucleic+acid+complexation+and+gene+transfer+efficiency+by+polyethylenimine.">Mixing-sequence-dependent nucleic acid complexation and gene transfer efficiency by polyethylenimine.</a> Biomater Sci. 3 (7), 1124-1133 (2015).</li><li>Liu, Y. M., Reineke, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(glycoamidoamine)s+for+gene+delivery:+Stability+of+polyplexes+and+efficacy+with+cardiomyoblast+cells.">Poly(glycoamidoamine)s for gene delivery: Stability of polyplexes and efficacy with cardiomyoblast cells.</a> Bioconjugate Chem. 17 (1), 101-108 (2006).</li><li>Schwanhausser, B., 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=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473 (7347), 337-342 (2011).</li><li>Greco, C. T., Muir, V. G., Epps, T. H., Sullivan, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+tuning+of+siRNA+dose+response+by+combining+mixed+polymer+nanocarriers+with+simple+kinetic+modeling.">Efficient tuning of siRNA dose response by combining mixed polymer nanocarriers with simple kinetic modeling.</a> Acta Biomater. 50, 407-416 (2017).</li></ol>

The authors declare that they have no competing financial interests.

There are a few steps in the method that are particularly critical. When formulating the nanocarriers, the order of component addition and mixing speed are two important parameters influencing efficacy39. This protocol requires that the cationic component, mPEG-b-P(APNBMA), is added to the anionic component, siRNA, in a dropwise fashion while vortexing. Depending on the total formulation volume, this mixing process takes 3-6 s. To test if the nanocarriers were formed properly, measure the...

Small interfering RNAs (siRNAs) mediate post-transcriptional gene silencing through a catalytic RNAi pathway that is highly specific, potent, and tailorable to virtually any target gene1. These promising characteristics have enabled siRNA therapeutics to advance in human clinical trials for the treatment of numerous diseases, including metastatic melanoma and hemophilia2,3. However, significant delivery issues persist that have hindered translation4. In particular, delivery vehicles must remain stable and protect siRNAs from extracellular degradation, yet also release the payload into the cytoplasm5. Furthermore, many RNAi applications require improved methods to regulate gene silencing in space and time6, which will reduce side effects in siRNA therapeutics7 and enable transformative advances in applications ranging from cell microarrays for drug discovery8 to modulation of cell responses in regenerative scaffolds9. These challenges highlight the need for new materials and methods to better control binding vs. release in siRNA nanocarriers.
One of the most promising strategies for controlling siRNA release and enhancing spatiotemporal regulation is the use of stimuli-responsive materials10. For example, a wide variety of biomaterials have been engineered with changeable nucleic acid binding affinity in response to altered redox potential or pH, or applied magnetic fields, ultrasound, or light11. Although many of these systems demonstrate improved control over nucleic acid activity, the use of light as a trigger is particularly advantageous due to its instantaneous temporal response, precise spatial resolution, and ease of tunability12. Moreover, the potential of photo-sensitive technologies for regulating gene expression has been demonstrated by state-of-the-art inducible promoter and optogenetic regulator systems; however, these systems suffer from numerous challenges including limited capacities to regulate endogenous genes, safety concerns such as immunogenicity, and difficulties in delivering multi-component assemblies13,14,15. Photo-responsive siRNA nanocarriers are ideally suited to overcome these drawbacks and provide a simpler and more robust approach to spatiotemporally modulate gene expression16,17,18. Unfortunately, methods to accurately predict the resulting protein knockdown response remain elusive.
A key challenge is that quantitative evaluations of siRNA release are rare19,20, and even when these evaluations are performed, they have not been coupled to analyses of siRNA/protein turnover dynamics. Both the amount of siRNA released and its persistence/lifetime are important determinants of the resulting gene silencing dynamics; hence, a lack of such information is a major disconnect that precludes accurate prediction of dose-response in RNAi21. Addressing this challenge would expedite the formulation of the appropriate structure-function relationships in nanocarriers and better inform biomaterial designs22. Furthermore, such approaches would enable development of more effective siRNA dosing protocols. In an attempt to understand the dynamic silencing response, several groups have investigated mathematical models of RNAi23,24,25. These frameworks were successful in providing insights into siRNA-mediated changes in gene expression and identifying rate-limiting steps26. However, these models have been applied only to commercial gene delivery systems (e.g., Lipofectamine and polyethylenimine (PEI)) that are not capable of controlled siRNA release, and the complexity of the models has severely limited their utility27. These shortcomings highlight an unmet need for new materials capable of precisely tunable siRNA release combined with streamlined and easy-to-use predictive kinetic models.
Our method addresses all of these challenges through the integration of a light-sensitive nanocarrier platform with coupled methods to quantify free siRNA and model RNAi dynamics. In particular, our platform&#39;s precisely controlled siRNA release28 is monitored by two complementary methods for accurately quantifying encapsulated vs. unbound siRNA. The experimental data from these assays are entered into a simple kinetic model to predict gene silencing efficiencies a priori29. Finally, the on/off nature of the nanocarriers is easily exploited to generate cell patterns in gene expression with spatial control on the cellular length scale. Thus, this method provides an easily adaptable method to control and predict gene silencing in a variety of applications that would benefit from spatiotemporal regulation of cell behavior.

<table><tbody><tr><td>siRNA</td><td>Sigma-Aldrich</td><td>SIC001</td><td>non-targeted, universal negative control</td></tr><tr><td>mPEG-b-P(APNBMA)</td><td>synthesized in our lab</td><td>N/A</td><td>photo-responsive polymer</td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-100</td><td></td></tr><tr><td>sodium dodecyl sulfate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>436143</td><td></td></tr><tr><td>rubber gasket</td><td>McMaster-Carr</td><td>3788T21</td><td>0.5 mL thick</td></tr><tr><td>UV laser&amp;nbsp;</td><td>Excelitas Technologies</td><td>Omnicure S2000</td><td>collimating lens and 365 nm filter used</td></tr><tr><td>agarose</td><td>Fisher Scientific</td><td>BP160-100</td><td></td></tr><tr><td>ethidium bromide</td><td>Fisher Scientific</td><td>BP1302-10</td><td></td></tr><tr><td>siRNA labelled with Dy547</td><td>GE Healthcare Dharmacon, Inc.</td><td>custom order</td><td>fluorophore conjugated to 5&amp;rsquo; end of sense strand</td></tr><tr><td>microscope slide</td><td>Fisher Scientific</td><td>12-550-A3</td><td>pre-cleaned glass</td></tr><tr><td>Secure-Seal Spacer</td><td>Life Technologies</td><td>S24735</td><td>double-sided adhesive</td></tr><tr><td>LSM 780&amp;nbsp;</td><td>Carl Zeiss</td><td>N/A</td><td>confocal microscope</td></tr><tr><td>ZEN 2010</td><td>Carl Zeiss</td><td>N/A</td><td>FCS analysis software</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>N/A</td><td>programming language</td></tr><tr><td>NIH/3T3 cells&amp;nbsp;</td><td>ATCC</td><td>ATCC CRL-1658</td><td></td></tr><tr><td>DMEM</td><td>Mediatech</td><td>10-013-CV</td><td>growth media</td></tr><tr><td>fetal bovine serum</td><td>Mediatech</td><td>35-011-CV</td><td>heat-inactivated</td></tr><tr><td>penicillin-streptomycin</td><td>Mediatech</td><td>&amp;nbsp;30-002-CI</td><td></td></tr><tr><td>6-well plates</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>Opti-MEM</td><td>Life Technologies</td><td>11058021</td><td>transfection media</td></tr></tbody></table>

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.

Following the formulation of the nanocarriers, siRNA release assays were conducted to inform the irradiation conditions to be used in the in vitro transfections. Various dosages of light were applied to determine the percent of siRNA that was released. The first assay used gel electrophoresis to separate the free siRNA molecules from the siRNA molecules still complexed/associated with the polymer. Nanocarriers that were not treated with light remained stable and did not release a...

Watch this Scientific Journal Video about Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers at JoVE.com

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

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 ...

predicting-gene-silencing-through-spatiotemporal-control-sirna

Research

JoVE Journal

Bioengineering

7.3K Views.  University of Delaware.  We presenteren een nieuwe methode die foto-responsieve blokcopolymeren gebruikt voor efficiëntere spatiotemporale controle van gen-stilzetting zonder detecteerbare off-target effecten. Bovendien kunnen veranderingen in genuitdrukking voorspeld worden door middel van straightforward siRNA release analyses en eenvoudige kinetische modellering. 

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

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

Medicine

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 ...

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

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 ...

Biology

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

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

Samenvatting

Meer video's verkennen