Name: predicting gene silencing through the spatiotemporal control of sirna release from photo-responsive polymeric nanocarriers
Uploaded: 2017-07-21T15:00:00
Description: Watch this Scientific Journal Video about Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers at JoVE.com

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) poly...

<ol>
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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.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">siRNA</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:145px;">SIC001</td>
			<td style="width:195px;">non-targeted, universal negative control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">mPEG-b-P(APNBMA)</td>
			<td style="width:157px;">synthesized in our lab</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">photo-responsive polymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">HEPES</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP310-100</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">sodium dodecyl sulfate&#160;</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:145px;">436143</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">rubber gasket</td>
			<td style="width:157px;">McMaster-Carr</td>
			<td style="width:145px;">3788T21</td>
			<td style="width:195px;">0.5 mL thick</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">UV laser&#160;</td>
			<td style="width:157px;">Excelitas Technologies</td>
			<td style="width:145px;">Omnicure S2000</td>
			<td style="width:195px;">collimating lens and 365 nm filter used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">agarose</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP160-100</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">ethidium bromide</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP1302-10</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">siRNA labelled with Dy547</td>
			<td style="width:157px;">GE Healthcare Dharmacon, Inc.</td>
			<td style="width:145px;">custom order</td>
			<td style="width:195px;">fluorophore conjugated to 5&#8217; end of sense strand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">microscope slide</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">12-550-A3</td>
			<td style="width:195px;">pre-cleaned glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Secure-Seal Spacer</td>
			<td style="width:157px;">Life Technologies</td>
			<td style="width:145px;">S24735</td>
			<td style="width:195px;">double-sided adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">LSM 780&#160;</td>
			<td style="width:157px;">Carl Zeiss</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">confocal microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">ZEN 2010</td>
			<td style="width:157px;">Carl Zeiss</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">FCS analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">MATLAB</td>
			<td style="width:157px;">MathWorks</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">programming language</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">NIH/3T3 cells&#160;</td>
			<td style="width:157px;">ATCC</td>
			<td style="width:145px;">ATCC CRL-1658</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">DMEM</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">10-013-CV</td>
			<td style="width:195px;">growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">fetal bovine serum</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">35-011-CV</td>
			<td style="width:195px;">heat-inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">penicillin-streptomycin</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">&#160;30-002-CI</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">6-well plates</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">08-772-1B</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Opti-MEM</td>
			<td style="width:157px;">Life Technologies</td>
			<td style="width:145px;">11058021</td>
			<td style="width:195px;">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 ...

The overall goal of these methods is to unable the prediction of small interfering RNA mediated gene silencing efficiencies and facilitate control over the resulting protein expression levels, in a spatial temporal manner. This method can elucidate structure function relationships and stimuli responsive delivery vehicles. Gaining better control over binding versus release, can unlock translatable platforms and drug discovery, as well as regenerative medicine technologies.The main advantages of this technique are the changes in gene expression can be controlled and predicted on the base as a simple, siRNA release assays and kinetic modeling. Though these methods employ novel photo-responsive polymers, this set of assays can be readily adapted to test the wide variety of other stimuli responsive systems. To begin, prepare a 32 micrograms per milliliter siRNA solution, by adding siRNA to a 20 micromolar HEPES solution at pH six.Then, prepare a polymer solution by adding dissolved mPEG block polyAPN BMA polymers to a 20 millimolar HEPES solution at pH six. To achieve a concentration of 220 micrograms per milliliter, this concentration will result in an N to P ratio of four, meaning there will be four times as many amine groups on the polymer as phosphate groups on siRNA. Next, add the polymer solution drop-wise to an equal volume of the siRNA solution, while gently mixing on a vortex machine.Continue to vortex for 30 seconds following polymer addition. Then, incubate the samples in the dark at room temperature for 30 minutes. Add the STS solution to each nanocarrier solution drop-wise while gently mixing on a vortex machine.Continue to vortex for 30 seconds following SDS edition. Centrifuge the samples at 3, 000 Gs for five seconds, then, incubate the samples in the dark at room temperature for 30 minutes. Calibrate and set a UV laser with a 365 nanometer filter to an intensity of 200 watts per square meter.Next, cut a hole into a rubber gasket, place the rubber gasket onto a washed and dried glass slide. Pipette the nanocarrier SDS solution onto the glass slide inside the hole of the rubber gasket. Load an excess of solution onto the glass slide, while avoiding contact with the rubber gasket.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. Attach binder clips to each side of the glass chamber to hold it close.Irradiate the samples for the desired length of time, using the UV laser with the 365 nanometer filter at an intensity of 200 watts per square meter. Remove the binder clips and open the chamber. Next, pipette 25 microliters of the irradiated nanocarrier SDS samples onto micro-centrifuge tubes.Incubate the solutions in the dark at room temperature for 30 minutes. Add five microliters of the loading buffer solution to 25 microliters of each nanocarrier SDS sample. Incubate the samples in the dark at room temperature for 10 minutes.Then, load 30 microliters of each nanocarrier SDS sample into a 2%agarose gel, pre-stained with ethidium bromide. Run the gel in the dark at 100 volts for 30 minutes. Image the gel, using a gel imaging system with ethidium bromide filters.Save the gel image files and proceed to bend intensity quantification as described in the text protocol. Remove the pieces of paper from a double sided adhesive spacer to expose the adhesive surface. Attach the spacer to a glass cover slip.Pipette the nanocarrier SDS solution onto the cover slip. Then, place a pre-washed glass slide on top of the cover slip with the hole from the adhesive spacer, centered around the sample. Push on the glass slide to ensure that the glass slide and cover slip are well attached and form a seal.Use a confocal microscope with a 40 times water immersive, aperchromal objective, having a numerical aperture of 1.2 for FCS measurements. Use the appropriate excitation laser channel to collect at least 30 measurements of 10 seconds each, per sample. Then, input the siRNA release data from FCS into a kinetic model to predict gene silencing responses, as detailed in the text protocol.To prepare in-vitro siRNA delivery, first add two milliliters of cell suspension to each well of the six well plate. Let the cells adhere and recover for 24 hours in the incubator. After washing the cells with phosphate buffered saline, prepare them for transvection by adding 1.5 milliliters of serum and antibiotic free transvection medium.Next, add 25 microliters of nanocarrier solution, containing 30 picomoles of siRNA tp each well. Gently pipette the medium up and down to mix. Place the cells in the incubator for three hours.Remove the transvection medium and wash each well with PBS. Then, add one milliliter of supplemented growth medium and place the cells in the incubator to recover for 30 minutes. To prepare the cells for treatment with a photo stimulus, remove the supplemented growth medium.Then, add one milliliter of transvection medium to each well. After calibrating the UV laser as described in the text protocol, place the cells on a hot plate set to 37 degree celsius. Remove the cover from the six well plate.Irradiate the cells from above the plate for the desired time, using the UV laser with a 365 nanometer filter at an intensity of 200 watts per square meter. Remove the transvection medium and add two milliliters of supplemented growth medium. Place the cells in incubator until further analysis.To prepare a photo mask that completely blocks 365 nanometer light and minimizes reflections, first cut, punch and/or machine the desired shape into the photo mask. Glue the photo mask to the bottom of the six well plate, with the pattern centered onto the well containing the cells and the anti-reflective side facing the plate. Set up two ring stands approximately 25 meters apart and detach 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. Irradiate the cells from below the sample for the desired time, using a UV laser with a 365 nanometer filter, at an intensity of 200 watts per square meter. Remove the transvection medium and add two milliliters of supplemented growth medium.Then, place the cells in incubator to recover for at least 24 hours. Image the cells using fluorescence microscopy as described previously. Shown here, is an example of simple kinetic modeling of gene silencing dynamics.The model was run to predict the concentrations of mRNA, protein and siRNA, as a function of time following photo-induced siRNA release. The relative amounts of siRNA released following different dosages of light, were inputted as the initial siRNA concentration in the kinetic model. The resulting model predictions for the protein expression levels were an agreement with the experimentally determined expression levels obtained through western blotting.For visualization, GFP expressing cells were treated with GFP targeting siRNA and an anular photo mask was placed on the plate prior to irradiation to generate a circular pattern. Cells that were protected from the light exhibited fluorescence intensity that were indistinguishable from control samples not treated with siRNA and light. However, nearly all cells within the circular pattern exhibited no GFP expression, indicating efficient siRNA release and gene knockdown.Moreover, the use of light is a trigger enabled gene expression, to be controlled on cellular length scales. In summary, this method enables the rapid determination of irradiation conditions to spatio-temporally control siRNA release and predict gene silencing a priori. Employing this protocol will help elucidate the structure function relationships governing nanocarrier stability and efficacy.This technique may enable applications in regenerative medicine that requires spatio-temporal control over gene expression. These formulations also are well suited to the treatment of localized disorders such as skin cancer, as well as dermatological disorders.

Research

JoVE Journal

Bioengineering

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