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 success for treating previously ‘undruggable’ 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 (< 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.
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 “undruggable” 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 – 200 nm diameter in size leverage EPR22, and smaller si-NPs (~20 – 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) – 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 ‘glow’ of fireflies27. Accordingly, mammalian cells transfected with the firefly luciferase gene produce a bioluminescent ‘glow’ 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.
1. Preparation and characterization of si-NPs
- si-NP preparation
- 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.
- Add siRNA (50 μM in diH2O) to result in N+:P- ratio of 10. Mix polymer and siRNA solutions thoroughly by pipetting and let incubate for 30 min. The N+:P- ratio represents the number of positively-charged amine groups on the polymer to the number of negatively-charged phosphate groups on the siRNA and is calculated by the formula below:
where, mol Pol is the molar amount of polymer, RU amine is the number of repeating units of positively-charged amines per polymer, mol siRNA is the molar amount of siRNA, and bp siRNA is the number of base pairs per siRNA molecule.
- Add a 5-fold excess of 10 mM phosphate buffer (pH 8.0) and mix gently either by pipetting or inverting the tube. To confirm that the final pH is neutral (~7.2-7.5), pipette 10 μL of si-NP solution onto pH test strips.
NOTE: Citric acid and phosphate buffers are prepared according to the Millipore Sigma Buffer Reference Center Charts.
- 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.
- Physicochemical characterization of si-NPs
- Record the size and surface charge of resulting si-NPs using dynamic light scattering (DLS). Prepare a DLS sample by filtering 1 mL of si-NPs (0.1 – 1.0 mg/mL) through 0.45 μm pore-size syringe filters into a square quartz or polystyrene cuvette. Record size and surface charge measurements using a DLS instrument according to the manufacturer’s specifications.
- Confirm the size and morphology of si-NPs by imaging analysis using transmission electron microscopy (TEM).
- Add 5 μL of si-NP solution at 1 mg/mL to TEM grids and incubate for 60 s. Blot dry for 3 s.
- Add 5 μL of 3% uranyl acetate solution and incubate for 20 s. Blot dry for 3 s. Dry grids overnight under desiccation.
- Image grids according to the protocol established for the specific microscope to be used.
- Characterize the loading of siRNA in si-NPs at various N+:P- ratios using agarose gel retardation.
- To produce 2% agarose gel, add 2 g of electrophoresis grade agarose powder to 100 mL of 1x TAE (Tris-acetate-EDTA) buffer at pH 8.0. Stir to suspend agarose. Heat uncovered in microwave until all agarose is dissolved (1-3 min).
- Once cooled, add 5 μL of ethidium bromide (10 mg/mL in H2O), and mix well. Pour agarose into a gel tray and place comb to produce wells, letting dry for 30 min. Carefully remove comb to leave behind loading wells, and fill the gel tray to the max fill line with 1x TAE buffer.
- Generate si-NPs (according to the procedure above) at 0, 1, 2, 5, 7, 10, 20, and 40 N+:P- ratios. Place 2 μL aliquots of loading dye (no SDS and reducing agents) on paraffin film for each si-NP formulation. Mix 10 μL of si-NP solution with loading dye on paraffin film by pipette.
- Add si-NP/loading dye solutions to agarose gel wells. Run voltage source at 100 V for 35 min (or until samples have traversed 80% of gel length).
- Visualize siRNA bands on a UV transilluminator according to the manufacturer’s specifications.
- Confirm the size and morphology of si-NPs by imaging analysis using transmission electron microscopy (TEM).
2. Determining in vitro bioactivity of si-NPs
- Knockdown of the model gene luciferase
- Generate luciferase si-NPs (according to the procedure above) using luciferase siRNA and scrambled si-NPs using a scrambled siRNA sequence as a control. Formualte both si-NPs at the same final N+:P- ratio and at the optimum ratio identified by agarose gel retardation studies. Example siRNA sequences are included in the Table of Materials.
- Seed luciferase-expressing cells [MDA-MB-231/Luciferase (Bsd) stable cells] in 96-well black-walled plates at a density of 2,000 cells per well. Allow to adhere overnight in full media (DMEM, 10% FBS) in an incubator (37 °C, 5% CO2, 95% humidity).
- Dilute si-NPs into full serum media for a final volume of 100 μL per well and siRNA concentration of 100 nM. Treat cells for 24 h with si-NPs.
- After 24 h, remove treatments and replace media with full serum media containing 150 μg/mL D-luciferin. Incubate cells for 5 min before measuring luminescence on a plate reader or in vivo optical imaging system according to the manufacturer’s specifications.
- Replace luciferin-containing media with fresh, full serum media, and incubate 24 h more. Repeat the step above, removing media and replacing with full serum media containing 150 μg/mL D-luciferin, followed by a 5 min incubation prior to measuring luminescence at the 48 h timepoint.
- For longitudinal studies, maintain cells under sterile conditions while measuring luminescence. Continue to culture in fresh, full media between measurements after replacing luciferin-containing.
NOTE: The appropriate siRNA concentration will vary with different si-NPs and siRNA molecules. When using neutrally-charged polyplexes with an endosomolytic core (e.g., PEG-DB), 100 nM is typically well-tolerated by the cells and produces >75% luciferase knockdown. The mass ratio of PEG-DB to siRNA at 10 N+:P- ratio and 100 nM siRNA treatments (assuming 26 bp siRNA) is 23.3, i.e., add 23.3 ng of PEG-DB for every 1.0 ng of siRNA. For example, add 1.16 μL of 3.33 mg/mL polymer for 166.5 ng of siRNA to treat one well at 100 nM in a 96-well plate (100 mL media volume per well).
Some essential characteristics of effective si-NPs for in vivo siRNA delivery are the proper size (~20 – 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 size and surface charge upon formulation. DLS and TEM are used as complementary methods to observe si-NP size (both), polydispersity (DLS), and morphology (TEM). DLS measurements show that si-NP 1 has an average diameter of 35 nm, unimodal distribution indicated by the presence of a single peak, and low polydispersity indicated by a relatively narrow peak width (Figure 2A). TEM measurements confirm the size measurement from DLS, suggest the presence of a uniform population of si-NPs, and reveal the spherical morphology of the si-NPs (Figure 2C). DLS and TEM measurements of si-NP 2 reveal that it has undesirable size (>200 nm diameter) of average diameter 1,500 nm, a multimodal and polydisperse population, and aggregates in solution, forming no distinct particle morphology (Figure 2B,D). Both si-NPs 1 and 2 display neutral surface charge, indicated by near-zero mean zeta potential values (Figure 2E).
Loading efficiency of siRNA into si-NPs is characterized using an agarose gel retardation assay. Un-complexed siRNA migrates through the agarose gel and is visualized (due to binding of ethidium bromide) at the gel bottom. When siRNA is complexed with the polymer to form si-NPs, migration through the agarose gel is obstructed and siRNA is visualized at the gel top (or where it was loaded into wells). For PEG-DB-based si-NPs, complexation increases with increasing N+:P- ratios until full complexation is achieved at ~10-20 N+:P- ratio (Figure 3).
Luciferase is used as a model gene for the rapid assessment of si-NP gene silencing bioactivity. Bioluminescence measurements via a plate reader or in vivo optical imaging system allow for the rapid, high-throughput quantification of luciferase protein expression in well-plate format. This technique is considerably faster, cheaper, and less burdensome than analyzing gene silencing through traditional molecular analyses such as PCR (gene expression) and western blot (protein expression). By this method, luciferase-expressing cells are treated with luciferase si-NPs and scrambled si-NPs (as a control), and %Luciferase Activity is calculated by comparing luminescence signal to untreated cells. Bioactive luciferase si-NPs will exhibit significantly diminished %Luciferase Activity when compared directly to scrambled si-NP control, as can be seen for si-NP 1 in Figure 4. In contrast, luciferase si-NPs that do not reduce %Luciferase Activity compared to scrambled si-NP control (such as si-NP 2) are not considered bioactive (Figure 4). Often 48 h is the time of maximum gene silencing (Figure 4), but we have observed significant gene silencing at time points ranging from 24 h to 240 h post-treatment.
Figure 1. Polymer chemistry and si-NP schematic. (A) Chemical composition of PEG-DB diblock copolymer that composes si-NPs and endows neutral surface charge (PEG block) and pH-responsive behavior (DB block). (B) Self-assembly of si-NPs. At pH 4.0, DB block is water-soluble because DMAEMA tertiary amines are highly protonated. The positively-charged DB block electrostatically complexes negatively-charged siRNA molecules. The pH is then adjusted to ~7.4 by the addition of 5x pH 8.0 phosphate buffer, resulting in the “hydrophobization” of DB block and sequestration of siRNA and DB within the core of si-NPs. Please click here to view a larger version of this figure.
Figure 2. DLS and TEM characterization of si-NP size, surface charge, and morphology. (A, C) si-NP 1 represents a uniform sample with appropriate size (~50-100 nm diameter), whereas (B, D) si-NP 2 has formed undesirable, large and polydisperse aggregates. (E) Both si-NPs display near-neutral surface charge (zeta potential). Error bars represent standard error. Please click here to view a larger version of this figure.
Figure 3. Agarose gel retardation assay to assess si-NP siRNA loading efficiency. The disappearance of siRNA bands at the gel bottom indicate complexation of siRNA to polymer. Polymer-complexed siRNA is unable to migrate through the gel and is thus visualized at the top of the gel nearby the loading wells. As the N+:P- ratio is increased, siRNA complexation increases, as indicated by decreased intensity of the siRNA band at the gel bottom. Please click here to view a larger version of this figure.
Figure 4. Si-NP-mediated knockdown of the model gene Luciferase in Luciferase MDA-MB-231 cells. Luciferase activity at (A) 24 h and (B) 48 h post-treatment with either scrambled si-NPs or luciferase si-NPs at 10 N+:P- ratio and 100 nM siRNA dose. %Luciferase Activity is calculated by dividing the luminescence signal of treatment samples (scrambled and luciferase) by the luminescence signal of untreated cells. NOTE: si-NP 1 represents an effective formulation with gene silencing bioactivity, whereas si-NP 2 represents a formulation without gene silencing bioactivity. (*) indicates a statistically significant difference (p < 0.05) compared to the Scrambled group for a formulation at a given timepoint. Error bars represent standard error. Please click here to view a larger version of this figure.
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 is adjusted to neutral (pH 7.4), causing ‘hydrophobization’ of the DB block, micelle formation, and entrapment of the complexed siRNA within the core of the resulting polyplexes. These polyplexes are designed such that PEG forms a hydrophilic, inert shell around the DB/siRNA core, resulting in neutral surface charge that is necessary for systemic administration. The polymer chemistry and process of polyplex formation are outlined in Figure 1.
Rigorous physicochemical characterization of si-NPs is essential for determining whether formulations are appropriate for moving into biological testing. Size, surface charge, and particle shape/morphology are all important parameters that can impact biological performance. For systemic delivery to tumors, si-NP size should fall between 20 – 200 nm diameter22, and most recent studies suggest 20 – 50 nm diameter particles are ideal23. Surface charge should be neutral or slightly negative to minimize protein adsorption and opsonization28. Studies have investigated the connection between particle shape and pharmacokinetics/particle clearance, suggesting particles with high aspect ratio are desirable over spherical particles29,30,31. However, to date spherical particles are still most commonly employed, and to our knowledge, are the only particle shape to have been translated to human studies in oncology.
It is important to note that the uniform and consistent formation of polyplexes, such as the si-NPs presented here, is dependent upon a range of physicochemical parameters. We have found empirically that polyplex concentration, pH of the solution, and the ratio of polymer to siRNA (N+:P- ratio) all drastically impact polyplex formation. In our hands, siRNA concentration does not have a large impact on polyplex formation, but using polymer concentrations in excess of 3.33 mg/mL results in formation of large aggregates and inconsistent polyplex size. As these si-NPs are pH-responsive, a variety of problems can arise when the final pH of si-NP solutions is too acidic (< pH 7.2). Two ways to prevent these complications are to lyophilize polymers in pure diH2O so that there are no salts to change buffer composition upon dissolution and to re-make buffers frequently to prevent the “drifting” of buffer pH over time. To be cautious, the pH of buffers should be confirmed each time before making si-NPs, and the final pH of si-NP solutions should always be verified. Finally, the N+:P- ratio of si-NPs impacts siRNA loading efficiency and polyplex physicochemical parameters. There typically exists an ideal N+:P- ratio at which all siRNA is loaded but there is not an overabundance of un-complexed polymer which forms separate populations of micelles and increases cytotoxicity. For the si-NPs presented here, N+:P- 10-20 is the range in which optimum consistency and performance have been observed.
Luciferase reporter cell lines are used here for the rapid and high-throughput analysis of si-NP bioactivity. Luciferase reporter cell lines must be generated or purchased before starting bioactivity analysis, thus requiring an initial investment in time and expense. However, use of the luciferase reporter cells to assess gene silencing is faster, more amenable to high-throughput analysis, and cheaper over time than performing RT-PCR (to measure mRNA expression) or western blots (to measure protein expression). For instance, measuring luminescence in this assay typically takes just a few minutes as opposed to all-day or multi-day processes necessary to perform RT-PCR or western blots. Moreover, luminescence measurements can be conducted on well-plates, allowing for the simultaneous quantification of many samples (up to 96-well plates have been used by the authors). Lastly, D-luciferin is the only reagent needed above and beyond typical cell culture reagents, making the method much more affordable than RT-PCR or western blot. It should be noted however, that the luciferase reporter cell assay is limited to only assessing gene silencing of the model gene luciferase and cannot be used to measure gene silencing of other “therapeutic genes” of interest. Thus, the authors refer readers to several studies where RT-PCR and/or western blot has been used to assess gene silencing of therapeutic genes of interest24,32,33,34.
In addition to bioactivity, researchers should ideally consider a comprehensive gamut of in vitro biological tests to characterize si-NP performance. The luciferase assay described above can also be used to assess cell viability by comparing the luminescence signal of scrambled si-NP treated cells to untreated cells. Since the siRNA is a non-targeting sequence, any change in luminescence can be attributed to non-specific impact of the si-NPs on cell viability, and this effect is typically dependent on the polymer chemistry and molar amount. Techniques in flow cytometry and fluorescent microscopy can be used to assess cell uptake of si-NPs using fluorescently-labeled siRNAs, polymers, or both. Additionally, molecular techniques such as stem-loop PCR and Argonaut 2 immunoprecipitation can be used to precisely measure intracellular siRNA levels. Since siRNA is only bioactive if delivered to the cytosol, where it is loaded into RISC, si-NPs must trigger endosomal escape of siRNA once internalized by the cell. Assays used to experimentally measure endosomal escape include the red blood cell hemolysis assay (described in detail by Evans et al.35), fluorescent imaging of the colocalization of fluorescently labeled si-NP cargo and LysoTracker36, and most recently, fluorescent imaging of the recruitment of Galectin 8 to punctured endosomal vesicles37,38. Gathering data and synthesizing the results from in vitro experiments for cell viability, cell uptake, endosomal escape, and bioactivity provide the researcher complementary pieces of information from which to draw interpretations and garner mechanistic insight about si-NP (in)effectiveness.
In sum, nanoparticles capable of efficiently delivering siRNA have tremendous potential to treat disease. For example, in oncology, many genes that cause cancer progression, resistance to therapy, and metastasis are ‘undruggable’ by conventional pharmacology but could be treated using siRNA. However, prerequisite to their use in animal models of disease, siRNA nanomedicines (referred to here as si-NPs) require extensive physicochemical and biological characterization to ensure both their safety and effectiveness. To this end, methods have been described herein to produce and characterize si-NPs in vitro, by which the si-NPs can be assessed for suitability to carry forward into animal studies.
The authors disclose no potential conflicts of interest.
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).
|0.45 μm pore-size syringe filters||Thermo Fisher Scientific||F25133||17 mm diameter, PTFE membrane|
|0-14 pH test strips||Millipore Sigma||P4786|
|10x TAE buffer||Thermo Fisher Scientific/Invitrogen||AM9869|
|6-7.7 pH test strips||Millipore Sigma||P3536|
|96-well black walled plates||Corning||3603||Tissue-culture treated|
|Agarose Powder||Thermo Fisher Scientific/Invitrogen||16500|
|Citric acid monohydrate||Millipore Sigma||C1909|
|dibasic sodium phosphate dihydrate||Millipore Sigma||71643|
|D-luciferin||Thermo Fisher Scientific||88294||Monopotassium Salt|
|DMEM||Gibco||11995065||High glucose and pyruvate|
|ethidium bromide||Thermo Fisher Scientific/Invitrogen||15585011|
|loading dye||Thermo Fisher Scientific/Invitrogen||R0611|
|Luciferase siRNA||IDT||N/A||Antisense Strand Sequense: GAGGAGUUCAUUAUCAGUGC
Sense Strand Sequense: CAAUUGCACU
GAUAAUGAACUCCT*C* *DNA bases
|MDA-MB-231 / Luciferase (Bsd) stable cells||GenTarget Inc||SC059-Bsd||Luciferase-expressing cells sued to assess si-NP bioactivity|
|monobasic sodium phosphate monohydrate||Millipore Sigma||S9638|
|Scarmbled siRNA||IDT||N/A||Antisense Strand Sequense: AUACGCGUAUU
Sense Strand Sequense: CGUUAAUCGCGUAUAAUAC
GCGUA*T* *DNA bases
|square polystyrene cuvettes||Fisher Scientific||14-955-129||4.5 mL capacity|
|TEM grids||Ted Pella, Inc.||1GC50||PELCO Center-Marked Grids, 50 mesh, 3.0mm O.D., Copper|
|Trisodium citrate dihydrate||Millipore Sigma||S1804|
|uranyl acetate||Polysciences, Inc.||21447-25|
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