Dioxolane

Preparation of LNPs and siRNA encapsulation. The cationic lipids 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy- keto-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2″-(meth oxypolyethyleneglycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) were provided by Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized as described elsewhere.^{13 (link)} Cholesterol was purchased from Sigma (St Louis, MO). The anti-Factor VII Cy5-labeled siRNA was provided by Alnylam Pharmaceuticals (Cambridge, MA) and was used in a free form or was encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEG-S-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios). When required, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) was incorporated to assess cellular uptake, intracellular delivery, and biodistribution. For gene silencing experiments, anti-GAPDH siRNA (siGAPDH) was purchased from Dharmacon. The preformed vesicle method of siRNA encapsulation was employed as described elsewhere.^{6 (link),7 (link),13 (link)} Briefly, the lipid mixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) were dissolved in ethanol to a final lipid concentration of 10 mmol/l. This ethanol solution of lipid was added drop-wise to 50 mmol/l citrate, pH 4.0 to form multilamellar vesicles to produce a final concentration of 30% ethanol vol/vol. Large unilamellar vesicles were formed following extrusion of multilamellar vesicles through two stacked 80 nm Nuclepore polycarbonate filters using the Extruder (Northern Lipids, Vancouver, Canada). Encapsulation was achieved by adding siRNA dissolved at 2 mg/ml in 50 mmol/l citrate, pH 4.0 containing 30% ethanol vol/vol drop-wise to extruded preformed large unilamellar vesicles and incubation at 31 °C for 30 minutes with constant mixing to a final siRNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol and neutralization of formulation buffer were performed by dialysis against phosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2 regenerated cellulose dialysis membranes. Nanoparticle size distribution was determined by dynamic light scattering using a NICOMP 370 particle sizer, the vesicle/intensity modes, and Gaussian fitting (Nicomp Particle Sizing, Santa Barbara, CA). The particle size for all three LNP systems was ~70 nm in diameter. siRNA encapsulation efficiency was determined by removal of free siRNA using VivaPureD MiniH columns (Sartorius Stedim Biotech) from samples collected before and after dialysis. The encapsulated siRNA was then extracted from the eluted nanoparticles and quantified at 260 nm. siRNA to lipid ratio was determined by measurement of cholesterol content in vesicles using the Cholesterol E enzymatic assay from Wako Chemicals USA (Richmond, VA).
Preparation of large LNPs. A lipid premix solution (20.4 mg/ml total lipid concentration) was prepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at 50:10:38.5 molar ratios. Sodium acetate was then added to the lipid premix at a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids were subsequently hydrated by combining the mixture with 1.85 volumes of citrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting in spontaneous liposome formation in aqueous buffer containing 35% ethanol. The liposome solution was then incubated at 37 °C to allow for time-dependent increase in particle size. Aliquots were removed at various times during incubation to investigate changes in liposome size by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Once the desired particle size was achieved, an aqueous PEG lipid solution (stock = 10 mg/ml PEG-DMG in 35% (vol/vol) ethanol) was added to the liposome mixture to yield a final PEG molar concentration of 3.5% of total lipid. Upon addition of PEG-lipids, the liposomes maintained their size, effectively quenching further growth. siRNA was then added to the empty liposomes at an siRNA to total lipid ratio of approximately 1:10 (wt:wt), followed by incubation for 30 minutes at 37 °C to form loaded LNPs. The mixture was subsequently dialyzed overnight in PBS and filtered with a 0.45-µm syringe filter.
Cell culture of primary APCs. bmAPCs were prepared from BM progenitors by flushing 6- to 8-week-old C57BL/6 (Jackson Laboratory) mouse femurs and tibias with PBS. After 2 washes, BM-derived progenitor cells were cultured in 6-well plates at 2 × 10⁶ cells/ml in 5 ml in complete medium (RPMI 1640 supplemented with 2 mmol/l -glutamine, 100 U/ml penicillin, 100 µg streptomycin, 10% fetal calf serum, sodium pyruvate (1 mmol/l), nonessential aminoacids (1 mmol/l), and Hepes (10 mmol/l) all from Invitrogen supplemented with either 10% 1x L929 cell conditioned media for differentiation of MΦ or X63-Ag8-plasmacytoma-derived GM-CSF (gift from David Gray, University of Edinburgh, Edinburgh, UK) 80 µl/5 ml for DCs. On day 8, bmMΦ were stained with antibodies against I-Ab (AF6.120.1) and CD11b whereas bmDCs were stained with H-2Kb (AF6.88), I-Ab, CD11c (HL3) (Pharmingen), and DEC-205 to assess their purity and stage of maturation. All cells were cultured in a humidified atmosphere of 5% CO₂ at 37 °C. After 8 days of culture, more then 80% of cells expressed MΦ- and DC-specific markers, respectively, as determined by flow cytometry.
Silencing of APCs in vitro. The day prior to treatment, bmMΦ and bmDCs were washed and replenished with fresh media in the original plates. SiRNA against GAPDH (sense sequence: UGGCCAAGGUCAUCCAUGA) or negative control scrambled siRNA encapsulated in DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA LNPs was added on day 8 of culture at 1 and 5 µg/ml final concentration and incubated for 72 hours at 37 °C and 5% CO₂. In each experiment, one well was treated PBS and served as a negative control. The media was changed every other day while the concentration of siRNA was maintained at required concentrations. Following treatment, GAPDH and α-Tubulin protein expression was measured to assess the efficacy and specificity of formulated siRNA.
Western blotting: Adherent bmMΦ were washed with cold PBS in the original plate followed by addition of RIPA lysis buffer containing 1% Nonidet P-40 (NP-40), 120 mmol/l NaCl, 4 mmol/l MgCl₂, 20 mmol/l Tris-HCl pH 7.6, protease inhibitor cocktail (Roche) 1 mmol/l PMSF and NaF, and incubated for 45 minutes at 4 °C. Cells were harvested by scraping the adherent cells with a cell-scraper. Cell lysates were then transferred in to a microcentrifuge tube and spun at maximum speed for 10 minutes. Floating bmDCs were washed 1 x with cold PBS and lysed by resuspending and incubating in RIPA lysis buffer for 45 minutes. The lysates were spun for 10 minutes at maximum speed at 4 °C, and protein concentration was assessed using Bradford assay (Biorad). About 5 µg protein sample was resuspended in appropriate volume of 10% SDS-containing Laemmli buffer and boiled for 5 minutes at 100 °C. Samples were subsequently analyzed on a 10% gradient SDS-polyacrylamide gel electrophoresis (PAGE). The gel was transferred onto a nitrocellulose membrane (PallCorporation, Pensacola, FL), blocked with Odyssey Blocking Buffer (Li-cor Biosciences, Lincoln, NE) and costained with rabbit anti-GAPDH and mouse anti α-Tubulin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200 dilution overnight. IRDye 680CW donkey anti-rabbit IgG (H+L) and IRDye 800CW donkey anti-mouse IgG (H+L) (Li-Cor Biosciences) were used at 1:2000 dilution to detect the GAPDH and α-Tubulin, respectively, following 2-hour incubation in the dark. Blots were scanned on an Odyssey Infrared Imaging System to visualize the bands of interest. Images were acquired at medium intensity and resolution 3. Quantification of the data was performed using Photoshop 9.0 CS2 software. Absolute intensity for each selected sample band was obtained by multiplying the pixel value with the average intensity. The relative intensity was then calculated by dividing the absolute intensity of each sample band by the absolute intensity (pixel value × average intensity) of the standard (loading control).
Flow cytometry: The efficacy of formulated siRNA was also determined by intracellular staining and assessment with flow cytometry. For this, 72 hours following treatment with 5 µg/ml siGAPDH or scramble control formulated with the LNPs under study, bmMΦ and bmDCs were harvested, transferred into microcentrifuge tubes and spun at 12,000 for 4 minutes. Cells were washed twice with FACS staining buffer (2% FBS, 1 mmol/l EDTA, and 0.1% Na Azide) and resuspended in fixation buffer (paraformaldehyde 4%) for 20 minutes. Next, they were resuspended in permeabilization buffer (saponin 0.5%, Sigma Aldrich, St. Louis, MO), aliquoted, and costained with rabbit anti-GAPDH and mouse anti α-Tubulin antibodies at 1:20 dilution for 30 minutes at room temperature. After two washings with permeabilization buffer, cells were labeled with goat anti-rabbit IgG (H+L) Alexa-647 (Invitrogen) and goat anti-mouse IgG (H+L) PE-conjugated antibodies in the dark, to detect the GAPDH and α-Tubulin, respectively, in bmAPCs. Unstained and cells incubated with isotype controls were included in all experiments. Next, bmAPCs were washed extensively with permeabilization buffer and PBS, followed by resuspension in 300 µl FACS staining buffer prior to data acquisition. The LSRII flow cytometer (BD Biosciences, San Jose, CA) and FACSDiva software were used to measure protein expression following acquisition of 10,000 events. Data were analyzed by FlowJo software. GAPDH expression values were normalized against α-Tubulin and expressed as percent reduced fluorescence relative to scramble control that was considered 100%.
Intracellular delivery of siRNA by LNPs. On day 8, bmMΦ and bmDCs were transferred to 24-well plates, treated with 1 µg/ml siRNA-Cy5 in a free form or encapsulated in DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA LNPs, and maintained at 37 °C. Incubation was stopped by washing off the media and harvesting the cells after 2, 4, 6, 8, and 24 hours. Cells were also incubated with 0.5, 1, or 5 µg/ml Cy5-labeled siRNA for 24 hours to assess the dose-dependent intracellular delivery of siRNA. To investigate whether the intracellular bioavailability of siRNA in bmAPCs correlates with the cellular uptake of LNPs, in parallel scrambled siRNA was formulated with DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA labeled with spDiO and incubated with bmAPCs for the same time, at 10 µg/ml. Incubation was stopped in the same fashion after the same time intervals. Following treatment, bmAPCs were transferred into microcentrifuge tubes, spun at 12,000 rpm for 4 minutes and after three washes, they were resuspended in 300 µl FACS staining buffer. Samples were acquired using an LSRII flow cytometer to assess the presence of Cy5-labeled siRNA as well as spDiO-labeled LNPs intracellularly. The Cy5 fluorophore was excited using the HeNe 633 laser line and detected at the APC (FL 5) channel whereas the spDiO by the Argon laser and detected at FL1 channel. Data were acquired using FACSDiva software and analyzed by FlowJo software. Measurements were taken for 10,000 events. Fluorescence intensity was normalized against the untreated controls and was expressed as percent increase of mean fluorescence units.
Assessment of intracellular trafficking of LNP-siRNAs using ICM. To assess the intracellular distribution of encapsulated siRNA, a pulse-chase experiment was undertaken. BmMΦ and bmDCs were taken on day 8 of culture and grown on glass coverslips in 6-well plates until 70% confluent. Next, 2 µg/ml of Cy5-labeled siRNA free or encapsulated in DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA LNPs was added and incubated for 2 hours. Incubation was stopped by removing the media and washing the coverslips twice with 4 °C PBS. Fresh media was then added and cells were placed at 37 °C for 1, 2, 4, and 8 hours to assess the siRNA distribution over time. After each time point, cells were washed in cold PBS, fixed with 3% paraformaldehyde for 10 minutes, permeabilized with 0.1% saponin (Sigma, St Louis, MO), and stained with nuclear marker Propidium Iodide (Molecular Probes, Burlington, Canada) for 2 minutes to identify individual cells. After several washes, cover slips were then mounted onto microscope glass slides using slow fade medium (Molecular Probes) and examined under an immunofluorescent confocal microscope to assess the intracellular pattern of Cy5-labeled siRNA. Multiple images were captured with the ×60 objective following excitation with 633-nm laser line. ICM was also performed to visualize the presence of LNP-encapsulated Cy5-siRNA in endosomes and lysosomes over various incubation times. For this, after 0.5, 1, 2, 4, and 8 hours of siRNA chase and following fixation and permeabilization, cells were stained with rabbit anti-Early Endosomal Antigen 1 (EEA1, Sigma). After 16-hour incubation, another set of cell-containing glass slides were costained with EEA1 and goat anti-Lysosomal-Associated Membrane Protein 1 (LAMP1, Santa Cruz Biotechnology). Secondary Alexa-488-conjugated donkey anti-rabbit IgG (H+L) and donkey anti-goat IgG (H+L) Alexa-568 antibodies (Molecular Probes, OR, USA) were used to detect the endosomes and lysosomes, respectively. Isotype controls were used in all confocal microscopy experiments to confirm the specificity of antibody staining. All images were acquired using a Nikon-C1, TE2000-U immunofluorescent confocal microscope, and the EZ-C1 software. Fluorochromes were excited using the 488-nm, 568-nm, and 633-nm laser lines, and multiple images were captured using the ×60 objective.
Image quantification: For consistency, during the image analysis, the nuclei were shown in blue and the siRNA is shown in red. EZ-C1 3.20 FreeViewer software was used to quantify the intracellular presence and distribution of siRNA in primary bmAPCs. For this, multiple spots of interest were drawn within the cell areas of high fluorescence intensity corresponding to vesicular-like compartments and areas of low fluorescence corresponding to cell cytoplasm. The average fluorescence intensity was then obtained by subtracting the background, from the high and low intensity values of 5–10 cells in one microscopic field. A minimum of three images were examined for each treatment and time point. Data were normalized and expressed as mean (±SD) percent of punctate or diffuse pattern relative to total fluorescence intensity. Colocalization of two molecules was analyzed using ImageJ.1 to select single slices, and Adobe Photoshop 9.0 CS2 was used to merge images obtained from green, red, and blue channels. For consistency, endosomes, lysosomes, and the siRNA were shown in green, blue, and red, respectively. Colocalization of two different molecules was evaluated by the presence, intensity, and distribution of the yellow color (green + red) or purple (red + blue). For the quantification of colocalization following dual staining, a total of ~50 cells were examined. Absolute fluorescence intensity of green, red, blue and overlapping yellow and purple colors was obtained by multiplying the pixel value with the average intensity. The relative fluorescence intensity of all individual colors was then expressed as percent of total green (endosomes) or blue (lysosomes) fluorescence. Analysis was performed for each of the 20 Z slices acquired, providing an overall quantification of colocalization for the entire cell.
In vivo gene silencing using DLinKC2-DMA LNPs. All experiments involving mice were performed in accordance with the requirements of Canadian Council and UBC Committee on Animal Care. To study the in vivo gene silencing properties of DLinKC2-DMA-formulated siRNA, on day 0, 6- to 8-week-old C57Bl6 (Charles River) triplicate mice received by tail vein 5 mg/kg siRNA targeting GAPDH (siGAPDH), siRNA against Factor VII (siFVII) as control, formulated with DLinKC2-DMA LNPs, or PBS. They were euthanized 4 days later to assess the gene silencing capacity of siGAPDH in PerC and spleen-derived APCs. Peritoneal cavity APCs were obtained following peritoneal irrigation with 10 ml RPMI containing 5% FBS and centrifugation at 1,500 for 10 minutes. Spleens were harvested, minced in small pieces, and digested in 1 mg/ml collagenase D (Roche) and CD11b⁺ and CD11c⁺ cells were isolated ex vivo using magnetic beads as described previously.^{50 (link)} Cell isolates were aliquoted, and GAPDH and α-Tubulin protein expression was assessed by flow cytometry as described. Data were acquired using LSRII flow cytometer after gating 10,000 events from the F4-80⁺/CD11b⁺ for MΦ or CD11c^high for DCs and analyzed by FlowJo software. The other aliquot of spleen-derived APCs was span down, lysed, and protein expression was assessed by western blotting and quantified as described earlier.
Factor VII analysis, CD45 silencing assay and GFP Longevity of silencing. Both serum Factor VII silencing, CD45 surface protein level measurements and 5′-RACE assay were done as described earlier.^{11 (link)} GFP mice (n = 2) were injected i.v. with DLinKC2-DMA LNPs encapsulated GFP or Luc-specific siRNA at 3 mg/kg. Peritoneal cavity MΦ were collected after 3 days and GFP expression was analyzed by FACS on CD11b⁺ cells. Remaining cells were plated in 96-well plate and GFP gene expression was analyzed by QPCR after 7, 14, and 21 days of post-injection.
TNF-α determination in vitro and in vivo. Cytokine induction by MΦ treated with LNP-siRNA was also studied in vitro and in vivo. BmMΦ were aliquoted in 48-well plates and treated with 1 and 5 µg/ml siRNA encapsulated with LNPs under study. Control wells were treated with PBS or LPS 10 ng/ml. At 2 hours, Brefeldin A (Sigma) 10 µg/ml final was added to the wells, and plates were returned to incubator (37 °C) for further 15 hours. Next, cells were washed, fixed-permeabilized, and stained with CD11b-FITC to detect MΦ and anti-TNF-α PE antibody (Invitrogen) to quantify the presence of TNF-α intracellularly using flow cytometry. Mice were also injected with siRNA formulated with DLinKC2-DMA and 72 hours later; spleen MΦ were isolated, aliquoted, and stimulated with 5 ng/ml PMA and 1 µg/ml ionomycin (Sigma) for 5 hours followed by incubation with Brefeldin A at 2-hour incubation. Control wells treated with PBS or LPS were also included. After 15 hours at 37 °C, intracellular staining was performed as described to detect the TNF-α expression in MΦ using flow cytometry.
Statistical analysis. Student's t-test (paired, two-sample equal variance) was used to compare the siRNA efficacy following delivery with formulations under study as indicated. The difference was considered statistically significant when P < 0.05 (two-tailed distribution).
SUPPLEMENTARY MATERIALFigure S1. DLinDMA is more toxic to APCs than DLinDAP, DLinK-DMA and DLinKC2-DMA.
Figure S2. Loss of viable cells following treatment with LNPs is due to apoptosis.
Figure S3. Biodistribution of LNP-siRNA and intracellular delivery in tissue resident APCs, B and T-cells.
Figure S4. Animals treated with DLinKC2-DMA siRNA did not show weight loss.
Figure S5. Gene silencing in macrophages is RNAi mediated.
Figure S6. In vivo transfected DCs activate T-cells and migrate in lymph nodes.
Materials and Methods.

The metabolically stable analogue of PAF, carbamyl-PAF (cPAF), and the PAF receptor antagonists PCA-4248 (22 (link)), CV-3988 (23 (link)), and (±)trans-2,5-bis(3,4,5-trimethoxyphenyl)-1,3-dioxolane (hereafter referred to as dioxolane [24 ] were purchased from Biomol). Egg yolk phosphatidylcholine was purchased from Sigma-Aldrich. Dr. Peter Isakson (G.D. Searle & Co., St. Louis, MO) provided SC-236 the selective COX-2 inhibitor. Stock solutions of cPAF, PCA-4248, CV-3988, dioxolane, and SC-236 were prepared at 5 mM concentrations by dissolving each in a 50% DMSO/PBS buffer and diluted further in PBS before cell culture or injection into mice. A thin layer of phosphatidylcholine (5 mM in PBS) was spread into a polystyrene dish and irradiated with 200 J/m² UVB under an FS-40 sunlamp. Irradiated solutions of phosphatidylcholine are referred to as UV-PC. The spontaneously transformed mouse keratinocyte cell line PAM-212 was obtained from Dr. Stuart Yuspa (National Cancer Institute, Bethesda, MD).

Sixty milliliters of a toluene solution of 4-bromobenzaldehyde (5.0 g, 27 mmol), ethylene glycol (4.3 g, 54 mmol), and p-toluene sulfonic acid (0.17 g, 0.90 mmol) was refluxed with a Dean-Stark trap for 8 hours. The solution was neutralized with 50 ml of K₂CO₃ aqueous solution and then extracted with ethyl acetate (3 × 50 ml). The organic layer was dehydrated by Na₂SO₄, filtrated, and dried under reduced pressure. The orange crude oil was purified using silica gel column chromatography (chloroform/hexane = 1:1), giving a compound 2 as yellow oil. Yield: 5.24 g (85%). ¹H NMR (400 MHz, DMSO-d₆, TMS): δ = 7.57 to 7.64 (d, 2H), 7.35 to 7.45 (d, 2H), 5.73 (s, 1H), and 3.90 to 4.08 (m, 4H).

Freshwater clam (C. fluminea, FC) was obtained from LiChuan Agriculture Farm (Changhua, Taiwan). The extraction method underwent modification based on a previous study [17 (link)]. First, to prepare the ethanol extract of freshwater clam (FCE), freshwater clam tissue was cut into small pieces, homogenized in a blender, and extracted three times with ethanol. The resulting supernatant was filtered and concentrated using a rotary evaporator under vacuum and then subjected to freeze-drying. Second, to prepare the ethyl acetate-extracted FCE (EA-FCE), the freeze-dried powder was extracted with ethyl acetate (FCE: water: ethyl acetate weight ratio = 1:2:3) and then subjected to freeze-drying. Third, TNHD was isolated from the EA-FCE by silica gel column chromatography (8 × 70 cm and 3.5 × 40 cm, Merck 40–63 μm) and further purified using HPLC with a Phenomenex Luna C-18 column (5 mm, 250 mm × 10 mm). The sample extraction process is shown in Fig. S1. The fractions were screened for bioactivity using a cell model, with those that showed high inhibition ability on HepG2 cells chosen and purified to obtain trans-2-nonadecyl-4-(hydroxymethyl)-1,3-dioxolane (TNHD) (Fig. S2A; Fig. S2B). The structure of TNHD was identified with NMR (Fig. S3).