The authors are grateful to Drs. Craig Duvall and Rebecca Cook for access to data and lab resources for conducting this research. The authors are grateful to the Vanderbilt Institute for Nanoscale Science and Engineering (VINSE) for access to DLS and TEM (NSF EPS 1004083) instruments. The authors are grateful to the National Science Foundation for supporting the Graduate Research Fellowship Program (NSF#1445197). The authors are grateful to the National Institutes of Health for financial support (NIH R01 EB019409). The authors are grateful to the Department of Defense Congressionally Directed Medical Research program for financial support (DOD CDMRP OR130302).

1. Preparation and characterization of si-NPs
<ol>
	<li>si-NP preparation
	<ol>
		<li>Dissolve polymer in 10 mM citric acid buffer (pH 4.0) at 3.33 mg/mL. Polymer can first be dissolved at 10x concentration in ethanol to ensure dissolution. 
		NOTE: Polymer can be dissolved at lower concentrations, but use at concentrations above 3.33 mg/mL can prevent homogenous NP formation.</li>
		<li>Add siRNA (50 &#956;M in diH2O) to result in N+:P- ratio of 10. Mix polymer and siRNA solutions...

<ol>
	<li>Fire, 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=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391 (6669), 806-811 (1998).</li><li>Elbashir, S. 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The authors disclose no potential conflicts of interest.

The si-NPs described here are formed by electrostatic association of anionic siRNA and cationic polymers into polyion complexes (polyplexes). Electrostatic complexing of siRNA and the cationic DB block of PEG-DB polymers is facilitated by mixing at low pH (4.0). At pH 4.0, DMAEMA is highly protonated, and consequently the DB block is highly charged. This ensures that the polymers dissolve as unimers in solution as opposed to forming micelles and that DB complexes efficiently with siRNA. Subsequently, the pH of solution i...

Because siRNAs inhibit the translation of proteins from mRNA sequences, they can theoretically be used to drug all known pathologies1,2,3,4,5. However, the use of siRNA in medicine is limited by the comprehensively poor pharmacokinetic profile of siRNA molecules6,7. When injected intravenously, siRNAs are rapidly cleared through the kidneys and/or degraded by nucleases8,9. Due to its large size and negative charge, siRNA cannot enter cells or escape the endolysosomal pathway to access the RNA-Induced Silencing Complex (RISC) that resides in the cytosol10,11,12,13. Thus, extensive effort has focused on the design and implementation of siRNA delivery strategies14. This effort has largely focused on the development of lipid- and polymer-based nanoparticles which package siRNA, protect it from clearance and degradation in vivo, and initiate cellular uptake and endosomal escape through ionizable, cationic amine groups. Many pre-clinical successes have been reported and most recently, the first clinical success has been reported for nanoparticle-based hepatic siRNA delivery to treat hereditary transthyretin-mediated (hATTR) amyloidosis15.
There are many cancer-causing genes that are currently &#8220;undruggable&#8221; by conventional pharmacology (i.e., small molecule drugs), motivating the design of polymeric siRNA nanoparticles (si-NPs) to treat cancer16. However, there are a separate set of design parameters that must be considered for non-hepatic siRNA delivery. The delivery system must shield the cationic charge of the polyplex which causes agglutination within the systemic circulation17,18,19. For tumor delivery, specifically, si-NP stability is essential to endow long circulation and thus increased accumulation within tumors via the enhanced permeability and retention (EPR) effect20,21. Moreover, control over si-NP size is essential since only nanoparticles approximately 20 &#8211; 200 nm diameter in size leverage EPR22, and smaller si-NPs (~20 &#8211; 50 nm diameter) exhibit improved tumor penetration over larger sized nanoparticles and microparticles23.
To address these additional design constraints for systemic tumor delivery of siRNA following intravenous administration, neutrally-charged, pH-responsive si-NPs have been developed (Figure 1)24. These si-NPs are PEGylated, or most recently, Zwitterionated25, for neutral surface charge and resistance to protein adsorption and opsonization in circulation. Since they cannot rely solely on cationic character to drive intracellular delivery, extremely efficient endosomal escape is imperative for achieving potent gene silencing. Accordingly, the core of these si-NPs is composed of a highly endosomolytic core which is inert at extracellular pH (7.4), but which is triggered in a switch-like manner in the acidified conditions of the endolysosomal pathway [pH 6.8 (early endosomes) &#8211; 5.0 (lysosomes)]. Lastly, a mixture of cationic and hydrophobic content within the core of si-NPs provide both electrostatic and van der Waals stabilization forces, improving stability of the si-NPs in blood compared to merely cationic systems.
The integration of many functions into a relatively simple design is possible using Reversible Addition-Fragmentation chain Transfer (RAFT) controlled polymerization to produce polymers with complex architecture and precise composition. To produce si-NPs with neutral surface charge, pH-responsiveness, and NP stability, RAFT is used to synthesize poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB; Figure 1A). PEG-DB is electrostatically complexed with siRNA, forming si-NPs with a PEG corona and DB/siRNA core (Figure 1B). PEG forms an inert, neutrally-charged hydrophilic layer on the si-NP corona. The DB block consists of a 50:50 molar ratio of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and butyl methacrylate (BMA). Cationic DMAEMA electrostatically complexes negatively-charged siRNA. BMA self-associates within the NP core by van der Waals interactions, increasing NP stability. Together, DMAEMA and BMA impart pH-dependent lipid bilayer-lytic behavior to the DB polymer block. At extracellular pH, the DB block is sequestered to the si-NP core and is inert to lipid bilayers. Under acidic conditions, such as those within the endolysosomal pathway, ionizable DMAEMA within the DB block facilitates the proton sponge effect, where endosomal buffering leads to osmotic swelling and rupture26. Additionally, hydrophobic BMA moieties within the DB block actively integrate into and lyse lipid bilayers, resulting in potent endosomolysis. Thus, siRNA is complexed with PEG-DB to form si-NPs that are neutrally-charged and highly stable at extracellular pH but which disrupt lipid bilayers at acidic pH, ensuring cytosolic delivery of the siRNA payload.
Herein are described the experimental procedures to produce si-NPs from PEG-DB. Methods to characterize the physicochemical parameters and bioactivity of si-NPs are presented and discussed. In order to rapidly assess si-NP bioactivity, luciferase is used as a model gene for knockdown studies. Firefly Luciferase is the protein responsible for the &#8216;glow&#8217; of fireflies27. Accordingly, mammalian cells transfected with the firefly luciferase gene produce a bioluminescent &#8216;glow&#8217; that can be captured using a luminometer to quantify levels of Luciferase expression. Here, we use Luciferase to assess bioactivity of si-NPs by delivering siRNA against Luciferase and quantifying the corresponding reduction in bioluminescence in Luciferase-expressing cells compared to cells that receive a scrambled siRNA.

<table>
	<tbody>
		<tr>
			<td>0.45 mm pore-size syringe filters</td>
			<td>Thermo Fisher Scientific</td>
			<td>F25133</td>
			<td>17 mm diameter, PTFE membrane</td>
		</tr>
		<tr>
			<td>0-14 pH test strips</td>
			<td>Millipore Sigma</td>
			<td>P4786</td>
			<td></td>
		</tr>
		<tr>
			<td>10x TAE buffer</td>
			<td>Thermo Fisher Scientific/Invitrogen</td>
			<td>AM9869</td>
			<td></td>
		</tr>
		<tr>
			<td>6-7.7 pH test strips</td>
			<td>Millipore Sigma</td>
			<td>P3536</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well black walled plates</td>
			<td>Corning</td>
			<td>3603</td>
			<td>Tissue-culture treated</td>
		</tr>
		<tr>
			<td>Agarose Powder</td>
			<td>Thermo Fisher Scientific/Invitrogen</td>
			<td>16500</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Millipore Sigma</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>dibasic sodium phosphate dihydrate</td>
			<td>Millipore Sigma</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin</td>
			<td>Thermo Fisher Scientific</td>
			<td>88294</td>
			<td>Monopotassium Salt</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995065</td>
			<td>High glucose and pyruvate</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Millipore Sigma</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>ethidium bromide</td>
			<td>Thermo Fisher Scientific/Invitrogen</td>
			<td>15585011</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>loading dye</td>
			<td>Thermo Fisher Scientific/Invitrogen</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase siRNA</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>Antisense Strand Sequense: GAGGAGUUCAUUAUCAGUGCAAUUGUU Sense Strand Sequense: CAAUUGCACUGAUAAUGAACUCCT*C* *DNA bases</td>
		</tr>
		<tr>
			<td>MDA-MB-231 / Luciferase (Bsd) stable cells</td>
			<td>GenTarget Inc</td>
			<td>SC059-Bsd</td>
			<td>Luciferase-expressing cells sued to assess si-NP bioactivity</td>
		</tr>
		<tr>
			<td>monobasic sodium phosphate monohydrate</td>
			<td>Millipore Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>Scarmbled siRNA</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>Antisense Strand Sequense: AUACGCGUAUUAUACGCGAUUAACGAC Sense Strand Sequense: CGUUAAUCGCGUAUAAUACGCGUA*T* *DNA bases</td>
		</tr>
		<tr>
			<td>square polystyrene cuvettes</td>
			<td>Fisher Scientific</td>
			<td>14-955-129</td>
			<td>4.5 mL capacity</td>
		</tr>
		<tr>
			<td>TEM grids</td>
			<td>Ted Pella, Inc.</td>
			<td>1GC50</td>
			<td>PELCO Center-Marked Grids, 50 mesh, 3.0mm O.D., Copper</td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Millipore Sigma</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>uranyl acetate</td>
			<td>Polysciences, Inc.</td>
			<td>21447-25</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Some essential characteristics of effective si-NPs for in vivo siRNA delivery are the proper size (~20 &#8211; 200 nm diameter), siRNA packaging, and gene silencing bioactivity. While this is not an exhaustive list (as addressed in the Discussion), these basic characteristics should be confirmed before considering further testing of a formulation.
Figure 2 illustrates the characterization of si-NP s...

Watch this Scientific Journal Video about Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery at JoVE.com

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

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 ...

The current protocol is significant because it allows researchers to produce siRNA loaded nanoparticles, and to adequately characterize these nanoparticles to ensure their suitability for administration to animals prior to subsequent studies. The advantage of this approach is to produce nanoparticles that increase the bioavailability of siRNA and to produce nanoparticles that are neutrally charged and thus suitable for systemic administration. This technique is impactful because siRNA nanoparticles can be applied to treat a myriad of diseases necessitating systemic administration including cancer, cardiovascular disease, and autoimmune disorders, among others.To begin, dissolve polymer in ethanol at a concentration of 33.3 milligrams per milliliter and stir to ensure dissolution. Then use a pipette to transfer polymer solution in 10 millimolar citric acid buffer to reach a concentration of 3.33 milligrams per milliliter. Add distilled water into a tube containing small interfering RNA to achieve a concentration of 50 micromolar.Mix the polymer and small interfering RNA solutions thoroughly in a tube by pipetting up and down to achieve a nitrogen to phosphate ratio of 10. Place the tube at ambient temperature for 30 minutes. Then add one milliliter of 10 millimolar phosphate buffer at pH eight to the tube to achieve a concentration of 0.28 milligrams per milliliter and mix gently either by pipetting or inverting the tube.To confirm that the final pH is neutral, around 7.2 to 7.5, pipette 10 microliters of the small interfering RNA nanoparticles solution onto the pH test strips. First, prepare a dynamic light scattering sample by filtering one milliliter of small interfering RNA nanoparticles at the concentration of 0.28 milligrams per milliliter through 0.45-micrometer pore size syringe filters into a square quartz or polystyrene cuvette. Then record size and surface charge measurements using a dynamic light scattering instrument according to the manufacturer's specifications.To confirm the size and morphology of small interfering RNA nanoparticles using transmission electron microscopy first add five microliters of filtered small interfering RNA nanoparticle solution at 0.28 milligram per milliliter to each TEM grid. Place the grids at ambient temperature for 60 seconds. Blot dry with filter paper for three seconds.Next, add five microliters of 3%uranyl acetate solution to each grid and incubate for 20 seconds. Blot dry for three seconds. Then place the grids in a desiccator to dry overnight before imaging under transmission electron microscopy.To perform agarose gel retardation, first add two grams of electrophoresis-grade agarose powder to 100 milliliters of 1X Tris-acetate-EDTA buffer at pH eight in a beaker to produce 2%agarose gel. Stir to suspend the agarose. Place the beaker uncovered in a microwave to heat for one to three minutes until all agarose is dissolved.Once cooled, add five microliters of ethidium bromide at the concentration of 10 milligrams per milliliter into the beaker and mix well. Then pour the agarose into a gel tray and place comb to produce wells. Let the gel dry for 30 minutes.Carefully remove the comb to leave behind loading wells, and pour 1X Tris-acetate-EDTA buffer into the gel tray to fill to the maximum fill line. Then place two microliter aliquots of loading dye with no SDS or reducing agents on paraffin film for each small interfering RNA nanoparticle formulation at different nitrogen to phosphate ratios. Pipette to mix 10 microliters of small interfering RNA nanoparticle solution with loading dye on paraffin film.Add the mixture to agarose gel wells. Plug the electrophoresis system to a power supply and run voltage source at 100 volts for 35 minutes or until samples have traversed 80%of gel length. Transfer the gel from the tray to a UV transilluminator and visualize the small interfering RNA bands on the UV transilluminator according to the manufacturer's specifications.Determine the optimum nitrogen to phosphate ratio based on the disappearance of small interfering RNA bands at the bottom of the gel. To knock down model gene luciferase, first seed luciferase-expressing cells in a 96-well black walled plate with DMEM in 10%FBS at a density of 2, 000 cells per well. Place the plate into an incubator overnight to allow the cells to adhere.In the morning remove media and add 10 microliters per well of small interfering RNA nanoparticles diluted into full serum media for a final small interfering RNA concentration of 100 nanomolar. Return the wells to the incubator for 24 hours to treat the cells with small interfering RNA nanoparticles. The next day, replace media with full serum media containing 150 micrograms per milliliter D-luciferin.Incubate the cells at room temperature for five minutes before measuring luminescence on a plate reader. Then repeat the media refreshment twice with fresh full serum media free of D-luciferin, followed by incubation for 24 hours. Replace the media with full serum media containing 150 milligrams per milliliter D-luciferin and incubate at room temperature for five minutes.Measure luminescence at the 48-hour time point. For longitudinal studies, leave lid on top of the well plate when outside of the biosafety cabinet to maintain cells under sterile conditions and continue the incubation after replacing luciferin-containing media with fresh full media. Dynamic light scattering measurements show that small interfering RNA nanoparticle one has an average diameter of 35 nanometers.The presence of a single peak with narrow peak width indicates unimodal distribution and low polydispersity. TEM measurements confirm the size measurement from dynamic light scattering suggests the presence of a uniform population of small interfering RNA nanoparticles and revealed the spherical morphology of the small interfering RNA nanoparticles. Too high concentration of polymer in the preparation step resulted for small interfering RNA nanoparticle two in undesirable diameter of greater than 200 nanometers, a multimodal and polydispersed population, and aggregates in solution forming no distinct particle morphology.Both small interfering RNA nanoparticles one and two display neutral surface charge indicated by near zero mean zeta potential values. Agarose gel retardation assay shows the disappearance of small interfering RNA bands at the gel bottom which indicates complexation of small interfering RNA to polymer. Polymer complexed small interfering RNA is unable to migrate through the gel and is thus visualized at the top of the gel nearby the loading wells.As the nitrogen to phosphate ratio is increased small interfering RNA complexation increases as indicated by decreased intensity of the small interfering RNA band at the gel bottom. Bioactive luciferase small interfering RNA nanoparticle one exhibits significantly diminished luciferase activity when compared to scrambled control. In contrast, luciferase small interfering RNA nanoparticle two is not considered bioactive.To produce consistent siRNA nanoparticles with desired physicochemical properties, one should use polymer solutions with appropriate concentrations and validate the pH of the buffer solutions at each step of the protocol. Using these methods in our research we've been able to produce siRNA nanoparticle-based therapies for previously undruggable cancer-causing genes and test the efficacy of these therapies in pre-clinical models of breast cancer.

preparation-neutrally-charged-ph-responsive-polymeric-nanoparticles

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Bioengineering

7.3K 조회수.  University of Mississippi. 현재 프로토콜은 연구원이 siRNA 로드 된 나노 입자를 생성하고 이러한 나노 입자를 적절하게 특성화하여 후속 연구 전에 동물에 대한 투여적합성을 보장하기 때문에 중요합니다. 이러한 접근법의 장점은 siRNA의 생체 이용률을 증가시키고 중하되어 전신 투여에 적합한 나노입자를 생성하는 나노입자를 생산하는 것이다. 이 기술은 siRNA 나노 입자암, 심혈관 질환 및 자가 면역 ?...