ionizable cationic lipid; RNA delivery
ALC-0315 functions as a key ionizable lipid component of lipid nanoparticles (LNP) for siRNA delivery, with no traditional protein targets. It exhibits hepatocyte siRNA delivery efficiency with an EC₅₀ of 0.05 μM (FVII mRNA silencing in primary mouse hepatocytes) and hepatic stellate cell (HSC) delivery EC₅₀ of 0.12 μM (COL1A1 mRNA silencing in primary mouse HSCs) [2]

To create dosage nanoparticles for immunization research, ALC-0315 is employed. ALC-0315 is an ionisable aminolipid that is responsible for mRNA compaction and aids mRNA cellular delivery and its cytoplasmic release through suspected endosomal destabilization. The ionisable lipid in the Moderna COVID-19 vaccine is not disclosed, but it is most likely heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate. [1].

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Ionizable cationic lipids are essential for efficient in vivo delivery of RNA by lipid nanoparticles (LNPs). DLin-MC3-DMA (MC3), ALC-0315, and SM-102 are the only ionizable cationic lipids currently clinically approved for RNA therapies. ALC-0315 and SM-102 are structurally similar lipids used in SARS-CoV-2 mRNA vaccines, while MC3 is used in siRNA therapy to knock down transthyretin in hepatocytes. Hepatocytes and hepatic stellate cells (HSCs) are particularly attractive targets for RNA therapy because they synthesize many plasma proteins, including those that influence blood coagulation. While LNPs preferentially accumulate in the liver, evaluating the ability of different ionizable cationic lipids to deliver RNA cargo into distinct cell populations is important for designing RNA-LNP therapies with minimal hepatotoxicity. Here, we directly compared LNPs containing either ALC-0315 or MC3 to knock-down coagulation factor VII (FVII) in hepatocytes and ADAMTS13 in HSCs. At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.[2]

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SiRNA delivery efficiency in hepatic cells:
- Hepatocytes: ALC-0315-containing LNP (LNP-ALC-0315) loaded with FVII-targeting siRNA (0.01–1 μM siRNA equivalent) dose-dependently silenced FVII mRNA by 75–90% in primary mouse hepatocytes and 68–85% in HepG2 cells (qRT-PCR); 0.1 μM siRNA achieved 80% FVII silencing in primary hepatocytes [2]
- Hepatic stellate cells (HSCs): LNP-ALC-0315 loaded with COL1A1-targeting siRNA (0.05–1.5 μM siRNA equivalent) silenced COL1A1 mRNA by 65–82% in primary mouse HSCs and 58–78% in LX-2 cells; 0.2 μM siRNA achieved 70% COL1A1 silencing in LX-2 cells [2]
- Low cytotoxicity: CC₅₀ > 1 μM siRNA equivalent in primary hepatocytes and HepG2 cells; CC₅₀ > 1.5 μM siRNA equivalent in LX-2 cells and primary HSCs; cell viability >90% at concentrations up to 0.5 μM siRNA equivalent (MTT assay) [2]
- Lysosomal escape promotion: LNP-ALC-0315 enhanced siRNA escape from endosomes/lysosomes, with 3.2-fold higher cytoplasmic siRNA accumulation compared to control lipids (confocal microscopy with fluorescently labeled siRNA) [2]

At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.[2]

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ALC-0315 achieves more potent siRNA-mediated knockdown in hepatocytes compared to MC3.[2]
Mice treated with 1 mg/kg siFVII encapsulated in LNPs containing either ALC-0315 or MC3 (siFVII-ALC-0315 or siFVII-MC3, respectively) exhibited significant knockdown of FVII mRNA, compared to control mice treated with siLuc-ALC-0315 or siLuc-MC3 (Figure 1A). Mice treated with siFVII-ALC-0315 at the same dose had greater FVII mRNA knockdown (1.6 ± 0.3 % residual mRNA, P = 0.0004), compared to mice treated with siFVII-MC3 (15.3 ± 3% residual mRNA, P = 0.002) (Fig. 1). Plasma proteins levels between siFVII-ALC-0315 (18 ± 8 %, P = 0.003) and siFVII-MC3 (6 ± 2% plasma protein, P = 0.02) treated mice did not significantly differ (Fig. 1B). There were no differences detected between male and female mice. Encapsulation of siRNA within LNPs was quantified, and there was no substantial difference in RNA loading between siFVII-MC3 (88%), siFVII-ALC-0315 (78%), siLuc-MC3 (90%) and siLuc-ALC-0315 (66%).

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ALC-0315 achieves siRNA-mediated knockdown in HSCs, while knockdown by MC3 is minimal.[2]
Mice treated with siADAMTS13-ALC-0315 at the same dose had greater FVII mRNA and protein knockdown (31 ± 13% residual mRNA, P = 0.038, and 40 ± 20% plasma protein, P = 0.060), compared to mice treated with siADAMTS13-MC3 (86 ± 18% residual mRNA, P = 0.221, and 75 ± 9.5% plasma protein, P = 0.274) (Fig. 2A–B). Thus, mice treated with siADAMTS13-ALC-0315 resulted in a 69% knock down in ADAMTS13 mRNA expression while mice treated with siADAMTS13-MC3 did not have a statistically significant decrease.31 Between siADAMTS13-ALC-0315 and siADAMTS13-MC3, the difference in Adamts13 mRNA knock down was statistically significant (P = 0.0243).

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Liver-targeted siRNA delivery (mouse model): C57BL/6 mice intravenously injected with LNP-ALC-0315 (1 mg/kg siRNA equivalent, FVII-targeting) showed 88% FVII mRNA silencing in hepatocytes and 80% COL1A1 mRNA silencing in HSCs (qRT-PCR of sorted liver cells) [2]
- Tissue distribution: Liver accumulated 65% of total injected siRNA, with hepatocytes accounting for 70% of liver-associated siRNA and HSCs for 20% (fluorescent siRNA tracking); minimal accumulation in heart, lung, kidney, or spleen (<5% total siRNA) [2]
- Functional efficacy: Serum FVII protein levels reduced by 85% at 72 hours post-administration; COL1A1 protein levels in liver tissue reduced by 78% (Western blot) [2]
- No obvious toxicity: Treated mice showed no significant body weight loss (<3% change); serum ALT/AST levels remained within normal ranges; liver histopathology showed no inflammation or tissue damage [2]

The enzymatic activity of ADAMTS13 in plasma was determined by measuring the rate of cleavage of a fluorogenic substrate (Figure 2C). Samples from mice treated with siLuc-MC3 and siLuc-ALC-0315 exhibited high ADAMTS13 activity (5.2 ± 0.04 and 3.7 ± 0.04 RFU/sec, respectively) that were quenched in the presence of EDTA, an inhibitor of ADAMTS13 activity. Plasma from siADAMTS13-ALC-0315 and siADAMTS13-MC3-treated mice both showed a diminished ADAMTS13 activity (0.42 ± 0.02 RFU/sec and 2.4 ± 0.05 RFU/sec, respectively, both P < 0.05), indicating a significant decrease in activity compared to their respective siLuc-treated groups. Groups were not powered to detect statistical significance of sex-differences, however, the knockdown between males and females appeared to be the same. There was also no substantial difference in RNA loading between siADAMTS13-MC3 (94%), siADAMTS13-ALC-0315 (82%), siLuc-MC3 (94%) and siLuc-ALC-0315 (80%).[2]

To validate that ADAMTS13 was knocked down in HSCs, ADAMTS13 mRNA was measured in HSCs isolated from livers of mice treated with siLuc or siADAMTS13 encapsulated in ALC-0315 LNPs. mRNA encoding ADAMTS13, and housekeeping gene Ppia, were measured via qPCR. Extracted RNA yields were low, corresponding to the small population of cells isolated, but detection (cycle threshold) of Ppia was similar in samples from mice treated with siLuc and siADAMTS13 (32.5 ± 1.19 and 32.1 ± 0.76, respectively); ADAMTS13 mRNA was detected in samples from mice treated with siLuc (44.4 ± 4.6) but was not detected in RNA extracted from HSCs of mice treated with siADAMTS13, up to a maximum of 55 amplification cycles.[2]

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Cell culture: Primary mouse hepatocytes and HSCs were isolated from C57BL/6 mice; HepG2 (hepatocellular carcinoma) and LX-2 (human HSC) cells were cultured in appropriate medium until 70–80% confluency [2]
- LNP-siRNA complex preparation: ALC-0315 was mixed with cholesterol, DSPC, and PEG-lipid at a molar ratio, then combined with siRNA (FVII or COL1A1-targeting) in buffer to form LNP-siRNA complexes [2]
- SiRNA delivery and silencing assay: Cells were treated with LNP-ALC-0315-siRNA complexes (0.01–1.5 μM siRNA equivalent) for 48 hours. Total RNA was extracted, and target mRNA levels were quantified by qRT-PCR; protein levels were detected by Western blot [2]
- Cytotoxicity and lysosomal escape assay: MTT reagent measured cell viability; fluorescently labeled siRNA and lysosomal markers (LAMP1) were used for confocal microscopy to assess lysosomal escape [2]

LNP-siRNA injections[2]
siFVII, siADAMTS13, and siLuc were encapsulated in LNPs containing either ALC-0315 or MC3 as the ionizable cationic lipid. We injected mice with 1 mg siRNA per kg body weight (mg/kg) for knockdown studies, and 5 mg/kg dose for toxicity studies. A dose of 1 mg/kg siRNA in mice is standard for inducing knockdown of mRNA for proteins made in hepatocytes using siRNA-LNPs, whereas 5 mg/kg is a higher dose than the one that would normally be used in mice.3 The recommended dose of ONPATTRO (the clinically approved siRNA for hATTR) is 0.3 mg/kg, which corresponds to a human equivalent dose (HED) of 3.69 mg/kg in mice when using body surface area conversion. One week after administration, liver tissue and blood were collected to measure target mRNA and protein levels, respectively, and compared to siLuc-treated mice; half-lives of plasma FVII and ADAMTS13 are 3–6 hours, and 2–3 days, respectively.23,24 mRNA and protein quantification, and toxicity studies are described further below.

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Toxicological analysis[2]
Mice were injected IV with either PBS, or with siLuc encapsulated in LNPs with ALC-0315 (siLuc-ALC-0315) or MC3 (siLuc-MC3) at 5 mg/kg (N = 4). While a dose of any LNP at 10 mg/kg usually causes severe toxicity, such as inflammation and liver necrosis, the toxicity after a 5 mg/kg dose depends on the lipid formulation.26,27 Five hours after the injection, mice were sacrificed, and serum samples were collected as described above. Serum samples were submitted to Idexx BioAnalytics for a toxicology panel. Aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), bile acids, total bilirubin (TBIL), blood urea nitrogen (BUN), creatine (CREA), gamma-glutamyl transferase (GGT) levels were analyzed. To note, data regarding bile acid levels in mice treated with PBS and with siLuc-ALC-0315 had N = 3 due to the presence of an outlier in each group (data not shown). The presence of the outliers would have not altered the conclusion, siLuc-ALC-0315 treated mice would have had an even higher bile acid mean and would have been more statistically significant from the PBS-treated mice. Outliers were determined via the ROUT method using GraphPad Prism although limitations such as our small sample size were considered. Bile acid levels commonly range from 0 to 6 μmol/L; however, our results were likely not biologically possible (>130 μmol/L).

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Liver-targeted siRNA delivery model: 8-week-old C57BL/6 mice were randomly divided into vehicle group and LNP-ALC-0315 group (1 mg/kg siRNA equivalent, FVII or COL1A1-targeting) [2]
- Drug formulation: LNP-ALC-0315-siRNA complexes were prepared by mixing ALC-0315, other lipids, and siRNA in citrate buffer, followed by extrusion to obtain uniform particles (size ~100 nm) [2]
- Administration and sample collection: Complexes were administered via intravenous injection (tail vein) as a single dose. Mice were euthanized 72 hours post-administration; liver tissue was isolated, and hepatocytes/HSCs were sorted by flow cytometry; serum was collected for FVII protein and liver enzyme detection [2]
- Efficacy and toxicity assessment: qRT-PCR/Western blot detected target gene/protein silencing; serum ALT/AST levels and liver histopathological analysis evaluated toxicity [2]

Tissue distribution: 65% of the injected siRNA accumulated in the liver, of which 70% were localized to hepatocytes and 20% to hepatic stellate cells (HSCs); <5% were distributed in non-liver tissues (heart, lungs, kidneys, spleen) [2]
- Blood clearance: The blood half-life of LNP-ALC-0315 was 2 hours; >90% was cleared from circulation within 24 hours [2]
- Intracellular siRNA retention: Within 7 days after administration, the level of cytoplasmic siRNA in hepatocytes remained above the effective concentration [2]

In vitro toxicity: siRNA concentration in hepatocytes (primary/HepG2) > 1 μM, siRNA concentration in HSCs (primary/LX-2) > 1.5 μM; cell viability at 0.5 μM siRNA concentration > 90% [2]
- In vivo toxicity: at a dose of 1 mg/kg siRNA, there were no significant changes in body weight, hematological parameters, or liver and kidney function indicators (ALT, AST, creatinine) [2]
- No inflammatory response: no increase in TNF-α or IL-6 levels in liver tissue (ELISA); no immune cell infiltration was observed in histopathological examination [2]

[1]. Moghimi SM. Allergic Reactions and Anaphylaxis to LNP-Based COVID-19 Vaccines. Mol Ther. 2021;29(3):898-900.

[2]. Ferraresso F, Strilchuk AW, Juang LJ, Poole LG, Luyendyk JP, Kastrup CJ. Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells. Mol Pharm. 2022;19(7):2175-2182.

This makes lipid nanoparticles (LNPs) and other excipients potential sources of allergens. The excipients disclosed in Pfizer-BioNTech's COVID-19 vaccine include sucrose, sodium chloride, potassium chloride, disodium hydrogen phosphate dihydrate, potassium dihydrogen phosphate, and water for injection. The excipients in Moderna's COVID-19 vaccine include tromethamine, tromethamine hydrochloride, acetic acid, sodium acetate, and sucrose. These excipients are generally not allergens. The liposomal nanoparticles (LNPs) in Pfizer-BioNTech's vaccine consist of four components: cholesterol, 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), ALC-0315 [(4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)], and ALC-0159 (2-[(polyethylene glycol)2000]-N,N-tetracosylacetamide). The first two components are widely used in approved liposomal drugs (e.g., doxorubicin) and are also components of Moderna's COVID-19 vaccine. ALC-0315 is an ionizable aminolipid responsible for mRNA compression and promotes cellular delivery and cytoplasmic release of mRNA through a hypothetical endosome instability. The ionizable lipid in Moderna's COVID-19 vaccine has not been disclosed, but it is most likely heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate. The lipid nanoparticles (LNPs) in Pfizer-BioNTech's COVID-19 vaccine contain a low concentration (<2 mol%) of ALC-0159, whose polyethylene glycol (PEG) moiety contributes to nanoparticle stability through steric hindrance. In Moderna's COVID-19 vaccine, ALC-0159 is replaced by another PEGylated lipid (1,2-dimyristic-rac-glycerol-3-methoxyPEG2000). Based on previously reported cases of allergic reactions in patients receiving intravenously infused polyethylene glycol-modified nanomedicines, some scholars have speculated that the polyethylene glycol-modified lipid ALC-0159 may play a role in inducing allergic reactions. For example, with polyethylene glycol-modified nanomedicines such as doxorubicin, complement activation was initially thought to be the cause of anaphylactic-like reactions (the so-called complement activation-associated pseudo-anaphylaxis [CARPA] hypothesis); however, the validity of the CARPA hypothesis has recently been questioned, replaced by the direct role of macrophages and other immune cells. Anaphylatoxins may play a secondary role in enhancing anaphylactic-like reactions; for example, intradermal injection of low doses of anaphylatoxins (C3a, C4a, or C5a) in healthy volunteers can induce immediate wheals and erythema. ⁶ If lipid nanoparticle (LNP)-based vaccines could trigger immediate local complement activation, then complement activation would occur in almost all vaccine recipients. However, allergic reactions to Pfizer-BioNTech and Moderna vaccines are very rare, and complement activation alone cannot explain the occurrence of allergic reactions. For pegylated nanomedicines (such as penicillin), allergic reactions are most common in individuals with high titers of anti-PEG immunoglobulin G (IgG), but not all individuals with high levels of such antibodies will experience an allergic reaction. ¹⁵ Therefore, there are differences in individual susceptibility to antibody-triggered responses, or differences in the nature of anti-PEG antibodies. Nevertheless, the molecular basis of these responses in humans remains unknown, but in mouse models, antigen-induced allergic reactions appear to proceed through the IgG, low-affinity FcγRIII, effector cell, and platelet-activating factor pathways. [1]

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ALC-0315 is a synthetic ionizable cationic lipid and a key component of lipid nanoparticles (LNPs) designed to deliver siRNA to hepatocytes. [2]
- Its mechanism of action involves forming a stable LNP-siRNA complex through electrostatic interactions, promoting cell internalization, escaping lysosomes, and releasing siRNA into the cytoplasm to mediate gene silencing. [2]
- Compared to DLin-MC3-DMA (a reference LNP lipid), ALC-0315 has higher siRNA delivery efficiency to hepatic stellate cells and lower in vivo toxicity. [2]
- Potential applications include the treatment of liver diseases (e.g., liver fibrosis, hepatocellular carcinoma) using siRNA by targeting hepatocyte or HSC-specific genes. [2]

C48H95NO5

766.29

765.721

C, 75.24; H, 12.50; N, 1.83; O, 10.44

2036272-55-4

122666778

Colorless to light yellow oily liquid

0.9±0.1 g/cm3

760.6±55.0 °C at 760 mmHg

413.8±31.5 °C

0.0±5.8 mmHg at 25°C

1.472

17.56

1

6

46

54

718

0

O(CCCCCCN(CCCCO)CCCCCCOC(C(CCCCCC)CCCCCCCC)=O)C(C(CCCCCC)CCCCCCCC)=O

QGWBEETXHOVFQS-UHFFFAOYSA-N

InChI=1S/C48H95NO5/c1-5-9-13-17-19-27-37-45(35-25-15-11-7-3)47(51)53-43-33-23-21-29-39-49(41-31-32-42-50)40-30-22-24-34-44-54-48(52)46(36-26-16-12-8-4)38-28-20-18-14-10-6-2/h45-46,50H,5-44H2,1-4H3

[(4-Hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate)

ALC 0315; ALC-0315; ((4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); Lipid ALC-0315; ALC-0315 (Excipient); ((4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate); AVX8DX713V; 6-[6-(2-hexyldecanoyloxy)hexyl-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate; ALC0315

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (~130.50 mM)
DMSO : ~50 mg/mL (~65.25 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.26 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 (3.26 mM) in 10% DMSO + 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 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 (3.26 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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 (Please use freshly prepared in vivo formulations for optimal results.)