Antibodies against cyclin A2 (mouse mAb), cyclin B1 (mouse mAb), cyclin D1 (mouse mAb), and cyclin E (mouse mAb) were purchased from Abcam, Inc. (Cambridge, MA). Silencer Select siRNAs (siRNA IDs for cyclin A2, B1, D1, and E are s2513, s2515, s229, and s2526, respectively), Silencer Select negative control siRNA, and the TaqMan® Fast Cells-to-CT™ Kit were purchased from Applied Biosystems™ by Life Technologies Corporation (Carlsbad, CA). Human Hep3B (HB-8064), human hepatocytes (CRL-11233), Eagle's Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), and 1X trypsin-EDTA solution (0.25% trypsin with 0.53 mM EDTA) were purchased from American Type Culture Collection (ATCC; Manassas, Virginia). 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoylsn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (18:1 PEG-2000 PE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). ABIL® EM 90 (cetyl PEG/PPG-10/1 dimethicone) was purchased from Evonik Industries (Essen, Germany). Hoechst 33342 (350/461), Alexa Fluor® 488 Antibody Labeling Kit (495/519), Alexa Fluor® 488 conjugate of annexin V (495/519), Alexa Fluor® 488-labeled mouse monoclonal antibody to BrdU (clone MoBU-1) (494/519), propidium iodide (535/617), Alexa Fluor® 647 carboxylic acid succinimidyl ester (650/668), SlowFade® Gold antifade reagent, Image-iT® FX signal enhancer, 1X Dulbecco's phosphate-buffered saline (D-PBS), bovine albumin fraction V solution (BSA, 7.5%), and Lipofectamine™ RNAiMAX were purchased from Invitrogen Life Sciences (Carlsbad, CA). BEGM Bullet Kits were purchased from Lonza Group Limited (Clonetics; Walkersville, MD). Amicon® Ultra-4 Centrifugal Filter Units (10 kDa MWCO) were purchased from Millipore (Billerica, MA). All peptides were synthesized by New England Peptide (Gardner, MA). Succinimidyl-[(N-maleimidopropionamido)-tetracosaethyleneglycol] ester (SM(PEG)24) was purchased from Pierce Protein Research Products (Thermo Fisher Scientific LSR; Rockford, IL). Ultra pure, EM-grade formaldehyde (16%, methanol-free) was purchased from Polysciences, Inc. (Warrington, PA). Absolute ethanol, hydrochloric acid (37%), tetraethyl orthosilicate (TEOS, 98%), 3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane (AEPTMS, technical grade), hexadecyltrimethylammonium bromide (CTAB, ≥ 99%), sodium dodecyl sulfate (SDS, ≥ 98.5%), Triton® X-100, hexadecane (≥ 99%), tert-butanol (≥ 99.5%), 2-mercaptoethanol (≥ 99.0%), DL-dithiothreitol ≥ (99.5%), dimethyl sulfoxide (≥ 99.9%), pH 5 citric acid buf fer, ethylenediaminetetraacetic acid (EDTA, 99.995%), sodium tetraborate (99%), glycine ≥ (99%), 5-bromo-2’-deoxyuridine (BrdU, ≥ 99%), goat serum, human epidermal growth factor, L-α-phosphatidylethanolamine, bovine fibronectin, bovine collagen type I, soybean trypsin inhibitor (≥ 98%), DMEM without phenol red, and Sephadex® G-200 were purchased from Sigma-Aldrich (St. Louis, MO). Holey carbon-coated copper TEM grids were purchased from SPI Supplies (West Chester, PA).
Cell Culture Conditions
Hep3B and hepatocytes were obtained from ATCC and grown per manufacturer’s instructions. Briefly, Hep3B was maintained in EMEM with 10% FBS. Hepatocytes were grown in flasks coated with BSA, fibronectin, and bovine collagen type I; the culture medium used was BEGM (gentamycin, amphotericin, and epinephrine were discarded from the BEGM Bullet kit) with 5 ng/mL epidermal growth factor, 70 ng/mL phosphatidylethanolamine, and 10% FBS. Cells were maintained at 37°C in a humidified atmosphere (air supplemented with 5% CO2) and passaged with 0.05% trypsin at a sub-cultivation ratio of 1:3.
Synthesis of Multimodal Silica Nanoparticles
The emulsion processing technique used to synthesize mesoporous silica nanoparticles with multimodal porosity has been described by Carroll et al.32
Briefly, 1.82 g of CTAB (soluble in the aqueous phase) was added to 20 g of deionized water, stirred at 40°C until dissolved, and allowed to cool to 25°C. 0.57 g of 1.0 N HCl, 5.2 g of TEOS, and 0.22 g of NaCl were added to the CTAB solution, and the resulting sol was stirred for 1 hour. An oil phase composed of hexadecane with 3 wt% ABIL®
EM 90 (a nonionic emulsifier soluble in the oil phase) was prepared. The precursor sol was combined with the oil phase (1:3 volumetric ratio of sol:oil) in a 1000-mL round-bottom flask, stirred vigorously for 2 minutes to promote formation of a water-in-oil emulsion, affixed to a rotary evaporator (R-205; Buchi Laboratory Equipment; Switzerland), and placed in an 80°C water bath for 30 minutes. The mixture was then boiled under a reduced pressure of 120 mbar (35 rpm for 3 hours) to remove the solvent. Particles were then centrifuged (Model Centra MP4R; International Equipment Company; Chattanooga, TN) at 3000 rpm for 20 minutes, and the supernatant was decanted. Finally, the particles were calcined at 500°C for 5 hours to remove surfactants and other excess organic matter.
To make unmodified particles more hydrophilic, they were treated with (i) 4% (v/v) ammonium hydroxide and 4% (v/v) hydrogen peroxide and (ii) 0.4 M HCl and 4% (v/v) hydrogen peroxide for 15 minutes at 80°C. Particles were then washed several times with water and re-suspended in 0.5 X D-PBS at a final concentration of 25 mg/mL. Mesoporous cores were modified with the amine-containing silane, AEPTMS, by adding 25 mg of calcined particles to 1 mL of 20% AEPTMS in absolute ethanol; the particles were incubated in AEPTMS for 4–6 hours at room temperature, centrifuged (5,000 rpm, 1 minute) to remove unreacted AEPTMS, and re-suspended in 1 mL of 0.5 X D-PBS. AEPTMS-modified particles were fluorescently-labeled by adding 5 µL of an amine-reactive fluorophore (Alexa Fluor® 647 carboxylic acid, succinimidyl ester; 1 mg/mL in DMSO) to 1 mL of particles; the particles were kept at room temperature for 2 hours prior to being centrifuged to remove unreacted dye. Fluorescently-labeled particles were stored in 0.5 X D-PBS at 4°C. Particles larger than ~400-nm in diameter were removed via size exclusion chromatography or differential centrifugation before cargo loading and liposome fusion; ~5% of the total mass of particles (mostly > 1-µm in diameter) was retained upon fractionation.
Characterization of Silica Nanoparticles
A 200 kV JEOL-2010 High Resolution Transmission Electron Microscope (JEOL, Ltd.; Carlsbad, CA) was used to image the mesoporous silica particles. Particles were dispersed in ethanol at a concentration of 5 mg/mL, and 4 µL of this solution was transferred onto a holey carbon-coated copper TEM grid (SPI Supplies; West Chester, PA). Excess liquid was wicked off using a Kim wipe, and the grid was allowed to dry before imaging. Dynamic light scattering of mesoporous silica nanoparticles, as well as cargo-loaded protocells and lipid nanoparticles, was performed using a Zetasizer Nano (Malvern; Worcestershire, United Kingdom). Samples were prepared by diluting 48 µL of silica particles (25 mg/mL) in 2.4 ml of 0.5 X D-PBS. Solutions were transferred to 1 mL polystyrene cuvettes (Sarstedt; Nümbrecht, Germany) for analysis. Zeta potential measurements were made using a Zetasizer Nano (Malvern; Worcestershire, United Kingdom). Silica particles were diluted 1:50 in 0.5 X D-PBS and transferred to 1-mL folded capillary cells (Malvern; Worcestershire, United Kingdom) for analysis. Nitrogen sorption was performed using an ASAP 2020 Surface Area and Porosity Analyzer (Micromeritics Instrument Corporation; Norcross, GA); surface area was determined using the Brunauer-Emmett-Teller (BET) model, and the cumulative pore volume plot was calculated from the adsorption branch of the isotherm using the Barrett-Joyner-Halenda (BJH) model. Pore size is defined as the Kelvin diameter plus the statistical thickness of the adsorbed film.
Liposome Fusion to Mesoporous Silica Nanoparticles
The procedure used to synthesize protocells has been described previously31,34,58,59
and will be mentioned only briefly. Lipids were ordered from Avanti Polar Lipids pre-dissolved in chloroform and stored at −20°C. Immediately prior to protocell synthesis, 2.5 mg of lipid was dried under a stream of nitrogen and placed in a vacuum oven (Model 1450M, VWR International, West Chester, PA) overnight to remove residual solvent. Lipids were re-hydrated in 0.5 X D-PBS at a concentration of 2.5 mg/mL and were passed through a 100-nm filter at least 10 times using a Mini-Extruder set (Avanti Polar Lipids, Inc.; Alabaster, AL). Resulting liposomes (~120-nm in diameter) were stored at 4°C for no more than one week. Mesoporous silica cores (25 mg/mL) were incubated with a 2- to 4-fold volumetric excess of liposomes for 30–90 minutes at room temperature. Protocells were stored in the presence of excess lipid for up to 1 month at 4°C. To remove excess lipid, protocells were centrifuged at 5,000 rpm for 1 minute, washed twice, and re-suspended in 0.5 X D-PBS.
Lipids were lyophilized together prior to rehydration and extrusion; for example 75 µL of DOPC (25 mg/mL), 5 µL of DOPE (25 mg/mL), 10 µL of cholesterol (75 mg/mL), and 10 µL of 18:1 PEG-2000 PE (25 mg/mL) were combined and dried to form liposomes composed of DOPC with 5 wt% DOPE, 30 wt% cholesterol, and 10 wt% PEG-2000. A DOPC:DOPE:cholesterol:18:1 PEG-2000 PE mass ratio of 55:5:30:10 was used to synthesize ‘DOPC protocells’, while a DOTAP:DOPE:cholesterol:18:1 PEG-2000 PE mass ratio of 55:5:30:10 was used to synthesize ‘DOTAP protocells’.
Conjugation of Peptides to the Supported Lipid Bilayer
SP94 and H5WYG peptides, synthesized with C-terminal cysteine residues, were conjugated to primary amines present in the head groups of PE using the heterobifunctional crosslinker, SM(PEG)24
, which is reactive toward sulfhydryl and amine moieties and possesses a 9.52-nm PEG spacer arm. Protocells were first incubated with a 10-fold molar excess of SM(PEG)24
for 2 hours at room temperature and centrifuged (1 minute at 5,000 rpm) to remove unreacted crosslinker. Activated protocells were then incubated with a 5-fold molar excess of SP94 for 2 hours at room temperature to attain a peptide density of 0.015 wt% (~6 peptides/protocell) and with a 500-fold molar excess of H5WYG for 4 hours at room temperature to attain a peptide density of 0.500 wt% (~240 peptides/protocell). Protocells were washed to remove free peptide, and average peptide density was determined by Tricine-SDS-PAGE, as described previously.31
Synthesis of siRNA-Loaded Protocells
AEPTMS-modified cores (25 mg/mL) were soaked in siRNA (250 µM in 1X D-PBS) for 2 hours at 4°C. Unencapsulated cargo was removed via
centrifugation at 5,000 rpm for 1 minute, and DOPC liposomes were immediately fused to cargo-loaded cores as described above. Unmodified cores were loaded with siRNA via
the synergistic mechanism previously described by us.34
Briefly, 25 µL of siRNA (1 mM) was added to 75 µL of silica nanoparticles (25 mg/mL). The solution was gently vortexed and incubated with 200 µL of DOTAP liposomes overnight at 4°C. Excess lipid and unencapsulated siRNA were removed via
centrifugation immediately before use.
Synthesis of siRNA-Loaded Lipid Nanoparticles
To prepare siRNA-loaded DOPC lipid nanoparticles (LNPs), DOPC, DOPE, cholesterol, and 18:1 PEG-2000 PE were first mixed in a 55:5:30:10 mass ratio, dried under a stream of nitrogen, and placed in a vacuum oven overnight to remove residual chloroform. The lipid film was then dissolved in tert-butanol and mixed 1:1 (v/v) with a siRNA solution (diluted in 10 mM Tris-HCl (pH 7.4) with 0.85% (w/v) NaCl and 0.25 M sucrose) such that the final DOPC:siRNA ratio was 10:1 (w/w). The mixture was vortexed, flash frozen in a bath of acetone and dry ice, and lyopholized. Immediately before use, the LNP preparation was hydrated with an isotonic sucrose solution (10 mM Tris-HCl (pH 7.4) with 0.85% (w/v) NaCl and 0.25 M sucrose) to a final siRNA concentration of 100 µg/mL; unencapsulated siRNA was removed via centrifugal-driven filtration (10 kDa MWCO).
We prepared siRNA-loaded DOTAP LNPs as described by Wu et al.
with minor modifications. We replaced PEGylated ceramide with 18:1 PEG-2000 PE and used a DOTAP:DOPE:cholesterol:PEG-2000 PE ratio of 55:5:30:10. We, additionally, dissolved lyopholized LNPs in 10 mM Tris-HCl (pH 7.4) with 0.85% (w/v) NaCl and 0.25 M sucrose to a final siRNA concentration of 100 µg/mL and removed unencapsulated siRNA using a centrifugal filtration device (10 kDa MWCO). LNPs were dissolved in 0.5 X D-PBS for zeta potential analysis.
To modify DOTAP LNPs with SP94 and H5WYG, they were first incubated with a 10-fold molar excess of SM(PEG)24 for 2 hours at room temperature; after removal of unreacted crosslinker via centrifugal-driven filtration (10 kDa MWCO), they were incubated with a 5-fold molar excess of SP94 and a 1000-fold molar excess of H5WYG for 2 hours at room temperature. Free peptide was removed using a centrifugal filtration device (10 kDa MWCO).
Determination of Cargo Capacities and Release Rates
The capacity of protocells and lipid nanoparticles (LNPs) for siRNA was determined by incubating 1 × 1010 particles in 1 wt% SDS (dissolved in D-PBS) for 24 hours and centrifuging the solutions to remove protocell cores and other debris. The concentration of siRNA in the supernatant was determined by comparing the absorbance at 260 nm to a standard curve.
The rate of siRNA release under neutral and acidic pH conditions was determined by suspending 1 × 1010 particles in 1 mL of a simulated body fluid (EMEM with 150 mM NaCl and 10% serum, pH 7.4) or citric acid buffer (pH 5.0) for various periods of time at 37°C. Particles were pelleted via centrifugation (5 minutes at 5,000 × g for protocells and 30 minutes at 15,000 × g for LNPs; Microfuge® 16 Centrifuge; Beckman-Coulter; Brea, CA). siRNA concentrations in the supernatant were determined using UV-visible spectroscopy, as described above. The concentration of released cargo was converted into a percentage of the cargo concentration that was initially encapsulated within 1010 particles.
Quantification of Cyclin A2, B1, D1, and E Protein Expression
To determine the concentration of siRNA necessary to silence 90% of cyclin A2, cyclin B1, cyclin D1, or cyclin E expression (IC90, see ), 1 × 106 Hep3B cells were exposed to various concentrations of siRNA loaded in SP94-targeted DOPC protocells for 48 hours at 37°C. Cells were then harvested by gentle shaking in 5 mM EDTA for 30 minutes at 37°C, centrifuged (1000 rpm, 1 minute) to remove excess particles, fixed with 3.7% formaldehyde (15 minutes at room temperature), and permeabilized with 0.2% Triton X-100 (5 minutes at room temperature); cells were then exposed to a 1:500 dilution of anti-cyclin A2, anti-cyclin B1, anti-cyclin D1, or anticyclin E, labeled using an Alexa Fluor® 488 Antibody Labeling Kit, for 1 hour at 37°C. Cells were washed three times and re-suspended in D-PBS for flow cytometry analysis (FACSCalibur). GraphPad Prism (GraphPad Software, Inc.; La Jolla, CA) was employed to calculate IC90 values from plots of log(siRNA concentration) versus mean fluorescence intensity; the initial protein concentration was taken to be the mean fluorescence intensity of antibody-labeled cells prior to treatment with siRNA-loaded protocells.
To determine the time-dependent decrease in cyclin A2, cyclin B1, cyclin D1, and cyclin E expression (see ), siRNA-loaded, SP94-targeted DOPC protocells were mixed with 1 × 106
Hep3B cells such that the final siRNA concentration was 125 pM; cells and protocells were incubated at 37°C for various periods of time, and resulting protein levels were determined via
immunofluorescence as described above. The same process was used to quantify cyclin levels in Hep3B treated with free siRNA, siRNA-loaded DOTAP LNPs, and SP94-targeted protocells loaded with Silencer
Select negative control siRNA (see the Supplementary Figures 2 and 3
); the total siRNA concentration was maintained at 125 pM in all time-dependent experiments.
The dose- and time-dependent decreases in cyclin A2 mRNA (, respectively) were determined by incubating Hep3B with SP94-targeted protocells loaded with the cyclin A2-specific siRNA as described above. Cells were washed three times with cold 1X PBS to remove excess protocells. mRNA was then isolated from cells and converted to cDNA using the TaqMan® Fast Cells-to-CT™ Kit. Quantitative PCR was performed by SeqWright, Inc. (Houston, TX).
To collect the data depicted in (left axis), a sufficient volume of siRNA-loaded, SP94-targeted DOPC protocells or DOTAP LNPs was added to 1 × 106 Hep3B or hepatocytes such that the final siRNA concentration was 125 pM. Samples were incubated at 37°C for 48 hours, and the resulting decrease in cyclin A2 expression was quantified as described above. To determine the values plotted in (right axis), 1 × 106 Hep3B cells were exposed to various concentrations (particles/mL) of siRNA-loaded, SP94-targeted DOPC protocells or DOTAP LNPs for 48 hours at 37°C; cyclin A2 expression was quantified using immunofluorescence, and the number of particles necessary to reduce cyclin A2 expression by 90% was calculated from a plot of particle concentration versus cyclin A2 concentration.
Cells depicted in were exposed to 10-fold excess of siRNA-loaded, SP94-targeted DOPC protocells with Alexa Fluor® 647-labeled cores for either 1 hour or 48 hours at 37°C. Cells were washed 3 times with D-PBS, labeled with Hoechst 33342 per manufacturer’s instructions, fixed with 3.7% formaldehyde (15 minutes at room temperature), permeabilized with 0.2% Triton X-100 (5 minutes at room temperature), and blocked with Image-iT® FX signal enhancer (30 minutes, room temperature). Cells were then exposed to Alexa Fluor® 488-labeled antibodies against cyclin A2, B1, D1, or E (diluted 1:500 in 1% BSA) overnight at 4°C, washed 3 times in D-PBS, and mounted with SlowFade® Gold.
Quantification of Growth Arrest
The numbers of proliferating and growth arrested Hep3B cells (, respectively) were determined by first exposing 1 × 106 cells to SP94-targeted, siRNA-loaded protocells for various periods of time at 37°C; protocells were loaded with a siRNA cocktail specific for cyclin A2, B1, D1, and E, and the total siRNA concentration was maintained at ~125 pM. Cells were then washed three times in 1X PBS to remove excess protocells. To determine the percentage of proliferating Hep3B, protocell-treated cells were incubated with 10 µM BrdU (in complete growth medium) for 12 hours at 37°C, harvested by gentle shaking in 5 mM EDTA for 30 minutes at 37°C, and fixed with 4% formaldehyde for 30 minutes at 4°C. Cells were then washed three times in 1X PBS with 0.1% Triton X-100; incubated in 1 N HCl for 10 minutes on ice; incubated in 2 N HCl for 10 minutes at room temperature and then 20 minutes at 37°C; incubated in 0.1 M borate for 12 minutes at room temperature; and washed three times in 1X PBS with 0.1% Triton X-100. Cells were blocked in 1X PBS with 0.1% Triton X-100, 1 M glycine, and 5% goat serum for one hour at room temperature and then incubated with an Alexa Fluor® 488-labeleled mouse monoclonal antibody to BrdU (1:100 dilution in 1X PBS with 1% BSA) overnight at 4°C. Cells were washed three times with 1X PBS, and the number of cells positive for BrdU incorporation was determined using a FACSCalibur flow cytometer. Cells were considered positive if their mean fluorescence intensities (MFI) were 100 fluorescence units (FU) greater than the MFI of unlabeled cells. To determine the percentage of G0/G1 and G2/M arrested Hep3B, protocell-treated cells were harvested by gentle shaking in 5 mM EDTA for 30 minutes at 37°C, incubated with 1 µg/mL of Hoechst 33342 for 15 minutes at 37°C, washed three times with 1X PBS, and immediately analyzed using a MoFlo High Performance Cell Sorter (Dako-Cytomation; Carpinteria, CA) equipped with Dako-Cytomation’s SUMMIT software, version 4.3.01. Cells were detected using a 488-nm Innova 90 laser (Coherent Inc.; Santa Clara, CA), and a gate was placed on the forward scatter-side scatter plot that excluded cellular debris. Hoechst 33342 was excited with a 355-nm Innova 90 laser, and emission intensity was collected in the FL-6 channel (450/65 filter/bandpass). Single cells were gated using width and area parameters; the area parameter histogram was used to determine the percentage of gated cells in G0/G1, S, and G2/M phases. Data were acquired with the SSC channel in log mode and all other channels in linear mode.
Quantification of Apoptosis
The time-dependent viability of Hep3B and hepatocytes (see ) exposed to siRNA-loaded, SP94-targeted protocells was determined by incubating 1 × 106 cells with 125 pM of siRNA for various periods of time at 37°C. Cells were harvested by gentle shaking in 5 mM EDTA for 30 minutes at 37°C, centrifuged (1000 rpm, 1 minute) to remove excess protocells and stained with Alexa Fluor® 488-labeled annexin V and propidium iodide per manufacturer’s instructions. The numbers of viable (double-negative) and non-viable (single- or double-positive) cells were determined via flow cytometry (FACSCalibur). Voltages were established using (1) untreated, unlabeled Hep3B (100% of cells were contained within the lower left quadrant, spanning from 100 to 102 fluorescence units on the FL-1 and FL-2 axes); (2) Hep3B transfected with the cyclin-specific siRNA cocktail using Lipofectamine™ RNAiMAX and singly stained with Alexa Fluor® 488-labeled annexin V (96% of cells were contained within the lower right quadrant, spanning from 102 to 104 FUs on the FL-1 axis and 100 to 102 FUs on the FL-2 axis); and (3) Hep3B transfected with the cyclin-specific siRNA cocktail using Lipofectamine™ RNAiMAX and singly stained with propidium iodide (98% of cells were contained within the upper right quadrant, spanning from 100 to 102 FUs on the FL-1 axis and 102 to 104 FUs on the FL-2 axis). Cells were transfected according to Invitrogen’s ‘reverse transfection’ protocol with an initial cell concentration of 5 × 105 (seeded in 60-mm plates), a final siRNA concentration of 50 nM, and a total incubation time of 72 hours.
To collect the data depicted in (left axis), a sufficient volume of siRNA-loaded, SP94-targeted DOPC protocells or DOTAP LNPs was added to 1 × 106 Hep3B or hepatocytes such that the final siRNA concentration was 125 pM. Samples were incubated at 37°C for 48 hours, and the number of apoptotic cells was determined as described above. To determine the values plotted in (right axis), 1 × 106 Hep3B cells were exposed to various concentrations (particles/mL) of siRNA-loaded, SP94-targeted DOPC protocells or DOTAP LNPs for 48 hours at 37°C; the number of apoptotic Hep3B was quantified using the annexin V/propidium iodide assay.
Cells shown in were exposed to a 10-fold excess of siRNA-loaded, SP94-targeted protocells with Alexa Fluor® 647-labeled cores for either 1 hour or 48 hours at 37°C. Cells were then washed 3 times with D-PBS, stained with Hoechst 33342, Alexa Fluor® 488-labeled annexin V, and propidium iodide per manufacturer’s instructions, fixed (3.7% formaldehyde for 10 minutes at room temperature), and mounted with SlowFade® Gold.
To collect the data depicted in Supplementary Figure 1
, 1 × 106
Hep3B cells were incubated with 1 × 109
AEPTMS-modified multimodal silica nanoparticles, DOPC protocells with AEPTMS-modified cores, or DOTAP LNPs, all loaded with Silencer
Select negative control siRNA for 48 hours at 37°C. Cells were then washed three times with 1X PBS to remove excess particles and stained with propidium iodide, per manufacturer’s instructions. Cells were immediately analyzed via
flow cytometry; cells were considered positive if their mean fluorescence intensities (MFI) were 100 fluorescence units (FU) greater than the MFI of unlabeled cells.
Flow Cytometry Equipment and Settings
For , , as well as Supplementary Figures 1–3
, cell samples were analyzed with a FACSCalibur flow cytometer (Becton Dickinson; Franklin Lakes, NJ) equipped with BD CellQuest™
software, version 5.2.1. Samples were acquired with the fsc channel in linear mode and all other channels in log mode. Events were triggered based upon forward light scatter, and, for data depicted in and Supplementary Figures 2 and 3
, a gate was placed on the forward scatter-side scatter plot that excluded cellular debris. Alexa Fluor®
488 was excited using the 488-nm laser source, and emission intensity was collected in the FL1 channel (530/30 filter/bandpass). Propidium iodide was excited using the 488-nm laser source, and emission intensity was collected in the FL2 channel (585/42). Mean fluorescence intensity was determined using FlowJo Software, version 6.4 (Tree Star, Inc.; Ashland, OR). All plots were generated using Sigma Plot, version 11.0 (Systat Software, Inc.; San Jose, CA).
Fluorescence Microscopy Equipment and Settings
Three- and four-color images were acquired using a Zeiss LSM510 META (Carl Zeiss MicroImaging, Inc.; Thornwood, NY) operated in Channel mode of the LSM510 software; a 40X, 1.4-NA oil immersion objective was employed in all imaging. Typical laser power settings were: 10% transmission for the 405-nm diode laser, 5% transmission (50% output) for the 488-nm Argon laser, 100% transmission for the 543-nm HeNe laser, and 80% transmission for the 633-nm HeNe laser. Gain and offset were adjusted for each channel to avoid saturation and were typically maintained at 500–700 and −0.1, respectively. 8-bit z-stacks with 1024 × 1024 resolution were acquired with a 0.7 to 0.9-µm optical slice. LSM510 and Zen 2009 Light Edition software were used to overlay channels and to create collapsed projections of z-stack images. All fluorescence images are collapsed projections.
For all microscopy experiments, cells were grown in culture flasks to 70–80% confluence, harvested (0.05% trypsin, 10 minutes), centrifuged at 4000 rpm for 2 minutes, and re-suspended in complete growth medium. 1 × 104 – 1 × 106 cells/mL were seeded on sterile coverslips (25-mm, No. 1.5) coated with 0.01% poly-L-lysine (150–300 kDa) and allowed to adhere for 4–24 hours at 37°C before being exposed to protocells. 48-hour samples were spun back onto coverslips using a Cytopro® Centrifuge, model 7620 (Wescor, Inc.; Logan, UT).