Ionizable cationic lipid; mRNA vaccine delivery
Erg-mediated Potassium Current (specifically human Erg1/Kv11.1 channel) [5]

To date, three ionizable cationic lipids have been approved for clinical use in RNA-based therapeutics: DLin-MC3-DMA (heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate), ALC-0315 (4-hydroxybutyl) azanediyl)bis (hexane-6,1-diyl)bis(2-hexyldecanoate), and SM-102(heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate). MC3 was designed for delivery of siRNA to hepatocytes to treat hereditary transthyretin amyloidosis (hATTR). ALC-0315 and SM-102 were designed for mRNA delivery, as they are the ionizable cationic lipids in the mRNA-based SARS-CoV-2 vaccines developed by Pfizer/BioNTech/Acuitas and Moderna, respectively.17,18 All three of these drug formulations use similar “helper” lipids and lipid molar ratios, approximately 50% ionizable cationic lipid, 10% DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine), 38.5% cholesterol, and 1.5% PEG-DMG (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000). MC3 contains major structural differences compared to ALC-0315 and SM-102, therefore differences in potency and toxicity may exist. Here, we report a head-to-head comparison of LNPs containing MC3 and ALC-0315, comparing their hepatotoxicity, and ability to deliver siRNA cargo in vivo to hepatocytes and HSCs. Due to the structural similarity to ALC-0315, SM-102 was not investigated in this study; both ionizable lipids exhibit similar branching, and the same functional groups (one hydroxy, one tertiary amine, two esters and only saturated hydrocarbons)[2].

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SM-102 concentration-dependently inhibited the amplitude of Erg-mediated potassium current in HEK293 cells stably expressing human Erg1 (Kv11.1) channels, with significant effects observed at concentrations ranging from 0.1 μM to 30 μM [5]
- SM-102 altered the gating kinetics of Erg1 channels: it shifted the voltage dependence of activation to more positive potentials, slowed the rate of activation and deactivation, and accelerated the rate of inactivation and reactivation [5]
- SM-102 induced prominent hysteresis in the Erg current-voltage relationship, which was concentration-dependent and more pronounced during depolarizing voltage ramps compared to hyperpolarizing ramps [5]

In this study, researchers evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. The study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection[4].

Protocols for mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles: [3]
Basic Protocol 1: Synthesis of mRNA by in vitro transcription and enzymatic capping and tailing
Basic Protocol 2: Encapsulation of mRNA into ionizable lipid nanoparticles
Alternate Protocol: Small-scale encapsulation of mRNA using preformed vesicles
Basic Protocol 3: Characterization and quality control of mRNA ionizable lipid nanoparticles.
For more details, please refer to https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.898

SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate) is an amino cationic lipid that has been tailored for the formation of lipid nanoparticles and it is one of the essential ingredients present in the ModernaTM COVID-19 vaccine. However, to what extent it may modify varying types of plasmalemmal ionic currents remains largely uncertain. In this study, we investigate the effects of SM-102 on ionic currents either in two types of endocrine cells (e.g., rat pituitary tumor (GH3) cells and mouse Leydig tumor (MA-10) cells) or in microglial (BV2) cells. Hyperpolarization-activated K+ currents in these cells bathed in high-K+, Ca2+-free extracellular solution were examined to assess the effects of SM-102 on the amplitude and hysteresis of the erg-mediated K+ current (IK(erg)). The SM-102 addition was effective at blocking IK(erg) in a concentration-dependent fashion with a half-maximal concentration (IC50) of 108 μM, a value which is similar to the KD value (i.e., 134 μM) required for its accentuation of deactivation time constant of the current. The hysteretic strength of IK(erg) in response to the long-lasting isosceles-triangular ramp pulse was effectively decreased in the presence of SM-102. Cell exposure to TurboFectinTM 8.0 (0.1%, v/v), a transfection reagent, was able to inhibit hyperpolarization-activated IK(erg) effectively with an increase in the deactivation time course of the current. Additionally, in GH3 cells dialyzed with spermine (30 μM), the IK(erg) amplitude progressively decreased; moreover, a further bath application of SM-102 (100 μM) or TurboFectin (0.1%) diminished the current magnitude further. In MA-10 Leydig cells, the IK(erg) was also blocked by the presence of SM-102 or TurboFectin. The IC50 value for SM-102-induced inhibition of IK(erg) in MA-10 cells was 98 μM. In BV2 microglial cells, the amplitude of the inwardly rectifying K+ current was inhibited by SM-102. Taken together, the presence of SM-102 concentration-dependently inhibited IK(erg) in endocrine cells (e.g., GH3 or MA-10 cells), and such action may contribute to their functional activities, assuming that similar in vivo findings exist[5].

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HEK293 cells stably expressing human Erg1 (Kv11.1) channels were cultured under standard conditions to reach confluency before experimentation [5]
- Whole-cell patch-clamp recordings were performed at room temperature (22-25 °C) in the voltage-clamp mode [5]
- Patch pipettes were pulled to achieve a resistance of 2-4 MΩ when filled with electrode internal solution, and the bath solution was continuously perfused during recordings [5]
- SM-102 was applied cumulatively to the bath solution at concentrations of 0.1 μM, 1 μM, 3 μM, 10 μM, and 30 μM, with a 5-minute equilibration period at each concentration before data collection [5]
- Erg currents were elicited by voltage protocols including step depolarizations and linear voltage ramps, and current traces were recorded and analyzed for amplitude, activation/inactivation kinetics, and hysteresis parameters [5]

Bioluminescence imaging[4]
\n\\n\\nTo detect the tissue distribution of SM-102-based LNPs formulation containing mRNA upon intravenous administration in mice, bioluminescence imaging (BLI) analysis was performed by using SM-102-based LNPs containing a firefly luciferase (FLuc) reporter mRNA. Briefly, 6–8 weeks old C57BL/6 mice were inoculated with 20 μg SM-102-based LNPs containing FLuc mRNA via the i.v. route. Six hours after injection, animals were given an intraperitoneal injection of luciferase substrate, and fluorescent signals were collected with an IVIS Spectrum instrument. The heart, liver, spleen, lung, and kidney tissues were collected, and the fluorescence signal of each tissue was detected by IVIS Spectrum instrument.[4]

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\\n\\nMouse experiments[4]
\n\\n\\nC57BL/6 mice and HBV transgenic mice were used. For AAV-HBV1.04 transduced mouse model, 6–8 weeks C57BL/6 male mice were injected with 5 × 1010 viral genome equivalents/mouse D genotype AAV-HBV1.04 through tail vein. After 10 days, the model was successfully built. To compare the efficiencies of CKK-E12- and SM-102-based nanoparticles, one dose of these two kinds of LNPs (3 mg/kg body weight) were injected into the mice respectively, and mice received PBS as control group. Then blood samples were collected at 2, 4 and 6 days after treatment. Livers were collected at 6 days after therapy. And in D genotype AAV-HBV1.04 transduced mice, we also evaluated SM-102-based LNPs encapsulating Cas9 mRNA and HBV targeting gRNAs effect with different doses. The mice received 3 or 1.5 mg/kg body weight SM-102-based LNPs encapsulating Cas9 mRNA and HBV targeting gRNAs. And mice received PBS as control group. The blood samples were collected at 2, 4 and 6 days after treatment. The livers were collected at 6 days after LNPs treatment. For HBV transgenic mice, 6 weeks old male mice were used to detect the therapeutic effect of LNPs. Two doses of nanoparticles encapsulating Cas9 mRNA with HBV or GFP targeting gRNAs were injected via i.v. into the mice at 1.5 mg/kg body weight. The blood samples were collected at day 2, 4, 11, 18 and 25 after therapy. The mice were sacrificed on day 25 post LNPs treatment. The blood was centrifuged at 1800 rpm for 15 min at 4 °C. Next, we collected the serum obtained to examine HBeAg, HBsAg, ALT and AST. The levels of HBV RNA, DNA and protein in the liver were determined. [4]

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\\n\\nTree shrew experiments[4]
\n\\n\\nThe tree shrews used in this study were originated from the Kunming Institute of Zoology, Chinese Academy of Science (Yunnan, China). Tacrolimus was dissolved in 5% DMSO, 40% PEG300, 5% Tween-80 and 50% saline. The resulting tacrolimus solution was injected via i.m. into the tree shrews at 0.08 mg/day/kg body weight in a total volume of 50 μl for the first 14 days. Tree shrews were inoculated i.p. with dexamethasone 10 mg/kg 2 days before and after HBV infection. Liposomal alendronate (CAS 121268-17-5) was injected via i.v. into tree shrews at 0.5 mg/kg the day before HBV infection. After Tacrolimus treated for 7 days, tree shrews were infected with HBV DNA positive human serum via the tail vein and the dose was adjusted to 106 copies/tree shrew. Three days later, tree shrews received 106 copies/tree shrew HBV via i.p. again. After the model successfully built, two doses of SM-102-based LNPs were injected into tree shrews at 1.5 mg/kg body weight for therapy. For control group, the tree shrews received LNPs encapsulating Cas9 mRNA and GFP targeting gRNAs. On day 2 and 4 after the treatment, the blood samples were collected. Tree shrews were sacrificed on day 5 post the nanoparticles treatment to determine the level of HBV RNA, DNA, protein and SMC5 in liver. And hepatitis B e antigen (HBeAg), HBsAg and HBV DNA levels in serum were also detected.

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\\n\\nSM-102-based LNP formulation[4]
\n\\n\\nLNPs were prepared by microfluidic techniques as reported previously (Mol. Ther., 26 (2018), pp. 1509-1519, 10.1016/j.ymthe.2018.03.010). In brief, SM-102 lipids were dissolved in ethanol at molar ratios of 50: 10: 38.5: 1.5 (ionizable lipid: DSPC: cholesterol: PEG-lipid). Chemically modified (2’ O-Methyl RNA/Phosphorothioated) HBV targeting gRNA and GFP targeting gRNA was synthesized. The RNA cargo (1: 1 wt ratio Cas9 mRNA: gRNA) was dissolved in 6.25 mM sodium acetate buffer (pH 5.0). Then, one volume of lipid mixture and three volume of RNA cargo were injected in to a NanoAssemblr microfluidic mixing device. The LNPs were dialyzed against PBS (pH 7.4) for overnight. The LNPs were passed through a 0.22 μm filter, and stored at 4 °C until use. RNA encapsulation efficiency was characterized by Ribogreen assay. The LNP size and ζ-potential were measured using a dynamic light scattering (DLS) technique.

[1]. A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates. Mol Ther. 2018 Jun 6;26(6):1509-1519.
[2]. Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells. Mol Pharm. 2022;19(7):2175-2182.
[3]. mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles. Curr Protoc. 2023;3(9):e898.
[4]. Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models. Antiviral Res . 2023 Jul:215:105618.
[5]. Effective Perturbations on the Amplitude and Hysteresis of Erg-Mediated Potassium Current Caused by 1-Octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6(undecyloxy)hexyl]amino]-octanoate (SM-102), a Cationic Lipid. Biomedicines . 2021 Oct 1;9(10):1367.

See Figure 1; the red arrow indicates the safe termination point. Basic Protocol 1: Synthesis of mRNA via in vitro transcription and enzymatic capping/tailing The entire protocol (including the preparation and evaluation of capped/tailed mRNA) takes 2 to 4 days. If overnight precipitation is planned, it takes 4 days. In vitro transcription, capping, and tailing reactions each take approximately half a day. Preparation for all reactions can be completed in about 1 hour, followed by the required incubation times: 2 hours for in vitro transcription and 1 hour for capping/tailing. mRNA precipitation requires a 30-minute centrifugation step, and RNA resuspending takes approximately 10 minutes. Quality assessment of mRNA using Nanodrop, agarose gel, and automated gel electrophoresis takes 1 hour. Note: More time is required when scaling up the experiment, especially when multiple or large mRNA precipitates need to be resuspended in nuclease-free water. Basic Protocol 2: Encapsulation of mRNA in iLNPs The preparation of the lipid solution can be performed before the formulation step. Otherwise, the entire encapsulation protocol must be completed on the same day. All reagents should be allowed to reach room temperature for 1 hour before use, and preparation and dilution in DPBS also require 1 hour. The time for the centrifugation concentration step depends on particle size, total sample volume, and the desired final volume. It typically takes 1 to 4 hours. The concentrated iLNP solution can be stored in a refrigerator until the required dilution is determined by RiboGreen assay.
Alternative: Small-scale mRNA encapsulation using pre-prepared vesicles
Same as Basic Protocol 2.
Basic Protocol 3: Characterization and Quality Control of mRNA iLNP
RiboGreen assay takes 1 to 2 hours, including the time for the kit to warm to room temperature after being removed from the refrigerator. DLS (particle size, PDI, zeta potential) analysis should be performed on the final diluted sample before biological evaluation. Sample preparation takes a few minutes, and analysis time varies depending on the instrument, but typically requires 5 to 10 minutes per sample.
The buffer used in the TNS assay must reach room temperature before use. This may take several hours depending on the aliquot size. The buffer can be refrigerated the night before the test to reduce the preheating time of the buffer. Each iLNP sample requires 40 aliquots in a 96-well plate, so each iLNP sample requires 10 to 15 minutes of plate preparation time and 10 minutes of plate reading time. The mRNA extraction step takes 30 minutes. For the method of analyzing the extracted mRNA using automated gel electrophoresis, see above (basic protocol 1). [3] There are 296 million people living with chronic hepatitis B virus (HBV) worldwide, which poses a serious health burden. The main challenge in curing HBV infection is that the source of persistent infection—free covalently closed circular DNA (cccDNA)—cannot be targeted and eliminated. In addition, although HBV DNA integration usually leads to the production of replication-defective transcripts, these transcripts are considered carcinogenic. Although several studies have evaluated the potential of gene-editing methods to target hepatitis B virus (HBV), previous in vivo studies have had limited relevance to real HBV infection because these models do not include HBV cccDNA and cannot simulate the complete HBV replication cycle under normal host immune system function. This study evaluated the effects of SM-102-based lipid nanoparticles (LNPs) co-delivering Cas9 mRNA and guide RNA (gRNA) in vivo on HBV cccDNA and integrated DNA in mice and higher animals. CRISPR nanoparticle treatment reduced the levels of HBcAg, HBsAg, and cccDNA in the liver of AAV-HBV1.04 transduced mice by 53%, 73%, and 64%, respectively. In HBV-infected tree shrews, the treatment reduced viral RNA by 70% and cccDNA by 35%. In HBV transgenic mice, HBV RNA inhibition reached 90%, and DNA inhibition reached 95%. CRISPR nanoparticle treatment was well-tolerated in both mice and tree shrews, with no observed increases in liver enzymes and extremely low off-target effects. Our study shows that SM-102-based CRISPR technology is safe and effective in targeting both free and integrated HBV DNA in vivo. SM-102-based lipid nanoparticle (LNP) delivery systems may be a potential therapeutic strategy against hepatitis B virus (HBV) infection. [4]

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SM-102, chemically named 1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate, is a cationic lipid. [5]
This compound exerts its biological effects through direct interaction with the Erg1 (Kv11.1) channel, a key mediator of cardiac repolarization and other physiological processes. [5]
The perturbation of Erg-mediated potassium currents by SM-102 suggests a potential impact on cardiac electrophysiology, as Erg channel dysfunction is associated with arrhythmias. [5]

C44H87NO5

710.1653

709.66

C, 74.42; H, 12.35; N, 1.97; O, 11.26

2089251-47-6

126697616

Colorless to light yellow oily liquid

15.5

1

6

43

50

686

0

O(C(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])O[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(=O)OC([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])=O)C([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H]

BGNVBNJYBVCBJH-UHFFFAOYSA-N

InChI=1S/C44H87NO5/c1-4-7-10-13-16-17-18-24-32-41-49-43(47)35-29-25-31-38-45(39-40-46)37-30-23-19-22-28-36-44(48)50-42(33-26-20-14-11-8-5-2)34-27-21-15-12-9-6-3/h42,46H,4-41H2,1-3H3

heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate

SM102; SM-102; heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; SM-102 (Excipient); T7OBQ65G2I; 1-Octylnonyl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate; heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate; Heptadecan-9-yl 8-[(2-Hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate; SM 102

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 (~140.81 mM)
DMSO : ~100 mg/mL (~140.81 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.52 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 (3.52 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 (3.52 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.)