Ionizable cationic lipid for siRNA delivery

Three primary regions comprise the structure of DLinDMA : the hydrocarbon chain, linker, and head group [1].

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LNP containing DLinKC2-DMA exhibits the most potent siRNA-mediated gene silencing in vitro in primary APCs. LNPs containing DLinKC2-DMA exhibit highest uptake into APCs. NPs containing DLinKC2-DMA exhibit the greatest cytoplasmic delivery of siRNA as determined by fluorescence microscopy. LNP siRNA systems deliver siRNA in APCs via endocytosis. NPs containing DLinKC2-DMA are relatively nontoxic to APCs [2].

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Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA , DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic [2].

In mice, the blood pharmacokinetic characteristics of DLinDMA are almost identical [1].

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Treatment with DLinKC2-DMA produces insignificant amount of TNF-α in vivo [2]
The potential of transfected MΦ to produce TNF-α in response to LNPs as a readout for cell stimulation by uptake was also studied. BmMΦ were incubated in vitro with different doses of siRNA formulated with the LNPs under study, or spleen MΦ of siRNA-DLinKC2-DMA-treated mice were isolated and TNF-α expression was assessed intracellularly by flow cytometry. Various expression levels of TNF-α were observed in bmMΦ following in vitro incubation with siRNA LNPs, and DLinDMA was the most potent cytokine inducer (Figure 8a). However, little amount of TNF-α was observed following DLinKC2-DMA treatment which showed levels similar to the least toxic LNP we know, the DLinDAP. Remarkably, MΦ transfected in vivo with DLinKC2-DMA expressed little amount of TNF-α (Figure 8b). Taken together the data indicate that there is a potential that LNP-siRNA complexes may induce cytokines in a dose-dependent manner and that the most efficient formulation DLinKC2-DMA produced insignificant amount of TNF-α in MΦ in vivo.

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In vivo-transfected DCs maintain their function to activate T cells and migrate in lymph nodes [2]
To test functional properties of LNP-treated APC, we tested the ability of in vivo-transfected DCs to activate T cells following antigen uptake (Supplementary Materials and Methods). For this, spleen DCs of mice injected with siRNA-DLinKC2-DMA were isolated, fed with ovalbumine, and their ability to activate the T-cell line B3Z35 was assessed and compared with the untreated DCs. Flow cytometry analysis revealed that there was no difference between control and in vivo-transfected DCs in their ability to activate T cells following cross-presentation of ovalbumine antigen (Supplementary Figure S6a). Furthermore, the migration potential of in vivo-transfected GFP+ DCs was also tested and compared with control DCs following injection in the foot pad of C57Bl6 mice. It was observed that almost equal number of cells migrated to inguinal LNs from the injection site in control and siRNA-DLinKC2-DMA-treated mice (Supplementary Figure S6b). Overall, the data indicate that there is essentially no functional impairment of DCs following their in vivo transfection with DLinKC2-DMA-formulated siRNA.

Silencing of APCs in vitro. [2]
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.

Intracellular delivery of siRNA by LNPs. [2]
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.

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Assessment of intracellular trafficking of LNP-siRNAs using ICM. [2]
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, and stained with nuclear marker Propidium Iodide for 2 minutes to identify individual cells. After several washes, cover slips were then mounted onto microscope glass slides using slow fade medium 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). After 16-hour incubation, another set of cell-containing glass slides were costained with EEA1 and goat anti-Lysosomal-Associated Membrane Protein 1 (LAMP1). Secondary Alexa-488-conjugated donkey anti-rabbit IgG (H+L) and donkey anti-goat IgG (H+L) Alexa-568 antibodies 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.

In vivo screening of cationic lipids for Factor VII activity. [1]
LNP-siRNA systems containing Factor VII siRNA were diluted to the appropriate concentrations in sterile PBS immediately before use and the formulations were administered intravenously through the lateral tail vein in a total volume of 10 ml/kg. After 24 h, animals were anesthetized with ketamine/xylazine and blood was collected by cardiac puncture and processed to serum (microtainer serum separator tubes; Becton Dickinson). Serum was tested immediately or stored at −70 °C for later analysis for Factor VII levels.

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In vivo nonhuman primate experiments. [1]
Cynomolgus monkeys (n = 3 per group) received either 0.03, 0.1, 0.3 or 1 mg/kg siTTR, or 1 mg/kg siApoB (used as control) formulated in KC2-SNALP as 15-min intravenous infusions (5 ml/kg) through the cephalic vein. Animals were euthanized 48 h after administration, and a 0.15–0.20 g sample of the left lateral lobe of the liver was collected and snap-frozen in liquid nitrogen. Prior studies have established uniformity of silencing activity throughout the liver6. TTR mRNA levels, relative to GAPDH mRNA levels, were determined in liver samples using a branched DNA assay. Clinical chemistry and hematology parameters were analyzed before and 48 h after administration.

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In vivo gene silencing using DLinKC2-DMA LNPs. [2]
To study the in vivo gene silencing properties of DLinKC2-DMA-formulated siRNA, on day 0, 6- to 8-week-old C57Bl6 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 and CD11b+ and CD11c+ cells were isolated ex vivo using magnetic beads as described previously.50 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 CD11chigh 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.

[1]. Rational design of cationic lipids for siRNA delivery. Nat Biotechnol. 2010 Feb;28(2):172-6.

[2]. Influence of Cationic Lipid Composition on Gene Silencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting CellsMol Ther. 2011 Oct 4;19(12):2186–2200.

We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.[1]

C41H77NO2

616.072

615.595

C, 79.93; H, 12.60; N, 2.27; O, 5.19

871258-12-7

871258-12-7;

11570822

Colorless to light yellow liquid

12.576

0

3

35

44

651

0

C(CN(C)C)(OCCCCCCCC/C=C\C/C=C\CCCCC)COCCCCCCCC/C=C\C/C=C\CCCCC

NFQBIAXADRDUGK-KWXKLSQISA-N

InChI=1S/C41H77NO2/c1-5-7-9-11-13-15-17-19-21-23-25-27-29-31-33-35-37-43-40-41(39-42(3)4)44-38-36-34-32-30-28-26-24-22-20-18-16-14-12-10-8-6-2/h13-16,19-22,41H,5-12,17-18,23-40H2,1-4H3/b15-13-,16-14-,21-19-,22-20-

N,N-Dimethyl-2,3-bis[(9Z,12Z)-octadeca-9,12-dienyloxy]propan-1-amine

DLinDMA; D-LinDMA; DLinDMA; 871258-12-7; 1,2-dilinoleyloxy-n,n-dimethyl-3-aminopropane; BD8S7LL258; N,N-dimethyl-2,3-bis[(9Z,12Z)-octadeca-9,12-dienoxy]propan-1-amine; N,N-Dimethyl-2,3-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propan-1-amine; 1,2-dilinoleyloxy-3-dimethylaminopropane; 1-Propanamine, N,N-dimethyl-2,3-bis((9Z,12Z)-9,12-octadecadien-1-yloxy)-; D-Lin-DMA

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Ethanol :≥ 100 mg/mL (~162.32 mM)
DMSO : ~100 mg/mL (~162.32 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.06 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.06 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.06 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (4.06 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: 2.5 mg/mL (4.06 mM) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: ≥ 2.5 mg/mL (4.06 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)